Contulakin-G, analogs thereof and uses therefor

ABSTRACT

The present invention is directed to contulakin-G (which is the native glycosylated peptide), a des-glycosylated contulakin-G (termed Thr 10 -contulakin-G), and derivatives thereof, to a cDNA clone encoding a precursor of this mature peptide and to a precursor peptide. The invention is further directed to the use of this peptide as a therapeutic for anti-seizure, anti-inflammatory, anti-shock, anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson&#39;s disease, gastrointestinal disorders, depressive states, cognitive dysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack, mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette&#39;s syndrome, Huntington&#39;s chorea, vascular leakage, anti-arteriosclerosis, vascular and vasodilation disorders, as well as neurological, neuropharmalogical and neuropsychopharmacological disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation application of U.S.patent application Ser. No. 10/067,857, filed on Feb. 8, 2002, which inturn is a continuation application of U.S. patent application Ser. No.09/420,797, filed on Oct. 19, 1999, now U.S. Pat. No. 6,369,193, eachincorporated herein by reference. The present invention is related toand claims priority under 35 U.S.C. §119(e) to U.S. provisional patentapplications Serial No. 60/105,015, filed on Oct. 20, 1998, Serial No.60/128,561, filed on Apr. 9, 1999 and Serial No. 60/130,661, filed onApr. 23, 1999, each incorporated herein by reference.

[0002] This invention was made with Government support under Grant No.GM-48677 awarded by the National Institutes of Health, Bethesda, Md. TheUnited States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The present invention is directed to contulakin-G (which is thenative glycosylated peptide), a des-glycosylated contulakin-G (termedThr₁₀-contulakin-G), and derivatives thereof, to a cDNA clone encoding aprecursor of this mature peptide and to a precursor peptide. Theinvention is further directed to the use of this peptide as atherapeutic for anti-seizure, anti-inflammatory, anti-shock,anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson'sdisease, gastrointestinal disorders, depressive states, cognitivedysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack,mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette'ssyndrome, Huntington's chorea, vascular leakage, anti-arteriosclerosis,vascular and vasodilation disorders, as well as neurological,neuropharmalogical and neuropsychopharmacological disorders.

[0004] The publications and other materials used herein to illuminatethe background of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are numerically referenced in thefollowing text and respectively grouped in the appended bibliography.

[0005] Mollusks of the genus Conus produce a venom that enables them tocarry out their unique predatory lifestyle. Prey are immobilized by thevenom that is injected by means of a highly specialized venom apparatus,a disposable hollow tooth that functions both in the manner of a harpoonand a hypodermic needle.

[0006] Few interactions between organisms are more striking than thosebetween a venomous animal and its envenomated victim. Venom may be usedas a primary weapon to capture prey or as a defense mechanism. Many ofthese venoms contain molecules directed to receptors and ion channels ofneuromuscular systems.

[0007] Several peptides isolated from Conus venoms have beencharacterized. These include the α-, μ- and ω-conotoxins which targetnicotinic acetylcholine receptors, muscle sodium channels, and neuronalcalcium channels, respectively (Olivera et al., 1985). Conopressins,which are vasopressin analogs, have also been identified (Cruz et al.1987). In addition, peptides named conantokins have been isolated fromConus geographus and Conus tulipa (Mena et al., 1990; Haack et al.,1990). These peptides have unusual age-dependent physiological effects:they induce a sleep-like state in mice younger than two weeks andhyperactive behavior in mice older than 3 weeks (Haack et al., 1990).The isolation, structure and activity of K-conotoxins are described inU.S. Pat. No. 5,633,347. Recently, peptides named contryphans containingD-tryptophan residues have been isolated from Conus radiatus (U.S. Ser.No. 09/061,026), and bromo-tryptophan conopeptides have been isolatedfrom Conus imperialis and Conus radiatus (U.S. Ser. No. 08/785,534).

[0008] It is desired to identify additional conopeptides havingactivities of the above conopeptides, as well as conotoxin peptideshaving additional activities.

SUMMARY OF THE INVENTION

[0009] The present invention is directed to contulakin-G (which is thenative glycosylated peptide), a des-glycosylated contulakin-G (termedThr₁₀-contulakin-G), and derivatives thereof, to a cDNA clone encoding aprecursor of this mature peptide and to a precursor peptide. Theinvention is further directed to the use of this peptide as atherapeutic for anti-seizure, anti-inflammatory, anti-shock,anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson'sdisease, gastrointestinal disorders, depressive states, cognitivedysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack,mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette'ssyndrome, Huntington's chorea, vascular leakage, anti-arteriosclerosis,vascular and vasodilation disorders, as well as neurological,neuropharmacological and neuropsychopharmacological disorders.

[0010] In one embodiment, the present invention is directed tocontulakin-G, contulakin-G propeptide and nucleic acids encoding thispeptide. The contulakin-G has the following formula:

Xaa₁-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Xaa₂-Tyr-Ile-Leu (SEQID NO: 1)

[0011] where Xaa₁ is pyro-Glu, Xaa₂ is proline or hydroxyproline andThr₁₀ is modified to contain an O-glycan. Xaa₂ is preferably proline. Inaccordance with the present invention, a glycan shall mean any N-, S- orO-linked mono-, di-, tri-, poly- or oligosaccharide that can be attachedto any hydroxy, amino or thiol group of natural or modified amino acidsby synthetic or enzymatic methodologies known in the art. Themonosaccharides making up the glycan can include D-allose, D-altrose,D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose,D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc),D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose. Thesesaccharides may be structurally modified as described herein, e.g., withone or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such assialic acid, including combinations thereof. The gylcan may also includesimilar polyhydroxy groups, such as D-penicillamine 2,5 and halogenatedderivatives thereof or polypropylene glycol derivatives. The glycosidiclinkage is beta and 1-4 or 1-3, preferably 1-3. The linkage between theglycan and the amino acid may be alpha or beta, preferably alpha and is1-. Preferred glycans are described further herein, with the mostpreferred glycan being Gal(β1→3)GalNAc(α1→).

[0012] In a second embodiment, the present invention is directed to ageneric contulakin-G having the following general formula,

Xaa₁-Xaa₂-Xaa₃-Xaa₃-Gly-Gly-Xaa₂-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Ile-Leu(SEQ ID NO:2),

[0013] where Xaa₁ is pyro-Glu, Glu, Gln or γ-carboxy-Glu; Xaa₂ is Ser,Thr or S-glycan modified Cys; Xaa₃ is Glu or γ-carboxy-Glu; Xaa₄ is Asn,N-glycan modified Asn or S-glycan modified Cys; Xaa₅ is Ala or Gly; Xaa₆is Thr, Ser, S-glycan modified Cys, Tyr or any unnatural hydroxycontaining amino acid (such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly,2,6-dimethyl-Tyr, 3-nitro-Tyr and 5-amino-Tyr); Xaa₇ is Lys,N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine,homoarginine or any unnatural basic amino acid (such asN-1-(2-pyrazolinyl)-Arg); Xaa₈ is Ala, Gly, Lys, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine, anyunnatural basic amino acid (such as N-1-(2-pyrazolinyl)-Arg) or X-Lyswhere X is (CH₂)_(n), phenyl, —(CH₂)_(m)—(CH═CH)—(CH₂)_(m)H or—(CH₂)_(m)—(C≡C)—(CH₂)_(m)H in which n is 1-4 and m is 0-2; Xaa₉ is Proor hydroxy-Pro; and Xaa₁₀ is Tyr, mono-iodo-Tyr, di-iodo-Tyr,O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, Trp, D-Trp, bromo-Trp,bromo-D-Trp, chloro-Trp, chloro-D-Trp, Phe, L-neo-Trp, any unnaturalaromatic amino acid (such as nitro-Phe, 4-substituted-Phe wherein thesubstituent is C₁-C₃ alkyl, carboxyl, hyrdroxymethyl, sulphomethyl,halo, phenyl, —CHO, —CN, —SO₃H and —NHAc, 2,6-dimethyl-Tyr and5-amino-Tyr). The C-terminus contains a free carboxyl group, is amidatedis acylated, contains a glycan or contains an aldehyde. It is preferredthat the C-terminus contains a free carboxyl. This peptide may furthercontain one or more glycans as described above. The glycans may occur atresidues 2, 7, 8, 10 and 16. The above and other unnatural basic aminoacids, unnatural hydroxy containing amino acids or unnatural aromaticamino acids are described in Building Block Index, Version 2.2,incorporated herein by reference, by and available from RSP Amino AcidAnalogues, Inc., Worcester, Mass.

[0014] In a third embodiment, the present invention is directed toanalogs of contulakin-G or the generic contulakin-G. These analogsinclude N-terminal truncations of contulakin-G or the genericcontulakin-G up to and including Thr₁₀. When the N-terminal truncationis through Thr₁₀, Lys₁₁ is N-glycosylated using a carboxylated modifiedlinker. This N-glycosylated Lys₁₁ can be represented as shown in FIG. 1(Toth et al., 1999), in which R₂, R₃ and R₄ are as described herein. Inthese truncations, it is preferred that the residue proximal to thetruncation is substituted with a glycosylated serine. Additional analogsinclude peptides in which Ser-O-glycan, Thr-O-glycan or Cys-S-glycan issubstituted for a residue at position 1-9.

[0015] In a fourth embodiment, the present invention is directed to usesof the peptides described herein as a therapeutic for anti-seizure,anti-inflammatory, anti-shock, anti-thrombus, hypotensive, analgesia,anti-psychotic, Parkinson's disease, gastrointestinal disorders,depressive states, cognitive dysfunction, anxiety, tardive dyskinesia,drug dependency, panic attack, mania, irritable bowel syndrome,diarrhea, ulcer, GI tumors, Tourette's syndrome, Huntington's chorea,vascular leakage, anti-arteriosclerosis, vascular and vasodilationdisorders, as well as neurological, neuropharmacological andneuropsychopharmacological disorders. In one aspect of this embodiment,analgesia is induced in a mammal using one of the peptides describedherein. In a second aspect of this embodiment, epilepsy or convulsionsare treated in a mammal. In a third aspect of this embodiment,schizophrenia is treated in an mammal. In a fourth aspect of thisembodiment, tardive dyskinesia and acute dystonic reactions are treatedin a mammal. In a fifth aspect of this embodiment, inflammation istreated in a mammal.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 shows the structure of an N-glycosylation of Lys using acarboxylated modified linker.

[0017]FIG. 2 shows the native O-glycan attached to Thr₁₀ ofcontulakin-G.

[0018]FIG. 3 shows analogs of the glycan which can be attached to one ormore residues of contulakin-G.

[0019]FIG. 4 shows the preferred core O-glycans (Van de Steen et al.,1998). Mucin type O-linked oligosaccharides are attached to Ser or Thr(or other hydroxylated residues of the present peptides) by a GalNAcresidue. The monosaccharide building blocks and the linkage attached tothis first GalNAc residue define the “core glycans,” of which eight havebeen identified. The type of glycosidic linkage (orientation andconnectivities) are defined for each core glycan.

[0020]FIG. 5 shows the purification of Contulakin-G. One gram of crudelyophilized venom from Conus geographus was extracted and applied on aSephadex G-25 column as previously described (Olivera et al., 1984).Three successive fractions containing paralytic and sleeper activities(Ve/Vo=1.37 to 1.41) were pooled, applied on a preparative reversedphase Vydac C₁₈ column and eluted with a gradient of acetonitrile in0.1% trifluroacetic acid. The component indicated by an arrow in panel Acaused wobbling and death when administered icv in mice.

[0021]FIG. 6 shows a nano-ESI MS/MS spectrum (m/z 1035 precursor) ofnative contulakin-G (286-1886 Da) (the MS/MS experiment is denoted usinga suggested shorthand (Mcluckey et al., 1991) where the closed circlerepresents m/z 1035 [M+2H]²⁺ precursor and the arrows are directedtowards the open circles which represent the fragments generated fromthe precursor). Above the spectrum, the structure of the glycoamino acidis represented where the arrows indicate 2 sites which lead to majorfragment ions observed in the MS/MS spectrum (Craig et al., 1993).

[0022] FIGS. 7A-7C show dose-response of CGX-1063 (Thr₁₀-contulakin-G)on spinally mediated (limb withdrawal) and supraspinally mediated(hindlimb lick) nociceptive behaviors elicited by noxious heat. Data areexpressed as seconds to response (FIGS. 7A and 7B) or to first fall(FIG. 7C). In FIG. 7A, latency to the first observable response afterplacement on a 50° C. hotplate is shown. FIG. 7B shows latency to thefirst hindpaw lick. FIG. 7C shows latency to first fall after placementon the accelerating rotorod (in FIGS. 7A-7C, n=3-10).

[0023] FIGS. 8A-8B show the effect of CGX-1063 on the nociceptiveresponse to persistent pain. In FIG. 8A, data are presented as theamount of time animals spent licking the formalin-injected hindpaw(n=7-10 animals/treatment group). Intrathecal CGX-1063 dose-dependentlydecreased the phase 2 nociceptive response in the formalin test comparedto intrathecal saline injected controls. FIG. 8B shows the latency tofirst fall from an accelerated rotorod immediately following theformalin test.

[0024]FIG. 9 shows paw withdrawal threshold to mechanical stimulationone week following partial sciatic nerve ligation. Data are presented asthe 50% withdrawal threshold in grams determined with calibrated vonFrey filaments (n=3-9 animals per group).

[0025] FIGS. 10A-10B show a comparison of CGX-1160 (contulakin-G),CGX-1063 and NT in the tail-flick test. Dose-response of the threecompounds is shown in FIG. 10A. FIG. 10B shows the duration of effect atthe highest doses tested for each compound (CGX-1160=100 pmol;CGX-1063=100 pmol; NT=10 nmol).

[0026] FIGS. 11A-11B show the effect of CGX-1160, CGX-1063 and NT onphase 1 (FIG. 11A) and phase 2 (FIG. 11B) of the formalin test. Allthree of the compounds dose-dependently reduced nociceptive behaviorfollowing i.pl. formalin. In phase 2 (FIG. 11B), CGX-1160 was 10 timesmore potent than CGX-1063, and 600-700 times more potent than NT.

[0027] FIGS. 12A-12C show effect of CGX-1160, CGX-1063 and NT on chronicinflammation-induced mechanical allodynia. Numbers in parenthesesindicate percentage of each corresponding control value. In FIG. 12A,CGX-1160 potently and dose-dependently reversed CFA-induced allodynia.In FIG. 12B, CGX-1063 reversed CFA-induced allodynia, but wasapproximately 100-fold less potent in this model than CGX-1160. In FIG.12C, NT reversed CFA-induced allodynia at 1,000 pmol, but not 100 pmol,approximately 10,000-fold less potent than CGX-1160.

[0028] FIGS. 13A-13B show locomotor impairing effects of CGX-1160,CGX-1063 and NT. FIG. 13A shows time to peak effect and duration ofeffect of the three compounds at the highest doses tested (approximately100 times the ED₅₀ in phase 2 of the formalin test). FIG. 13B showsdose-response of each compound on locomotor impairment.

[0029] FIGS. 14A-14C show dose-effect and time to peak effect andduration of locomotor impairment of CGX-1160, CGX-1063 and NT. FIG. 14Ashows that CGX-1160 caused long-lasting motor impairment only at doses100-fold or greater than its ED₅₀. FIG. 14B shows that CGX-1063 causedlong-last motor impairment at doses 10-fold or greater than its ED₅₀.FIG. 14C shows that NT caused long-last motor impairment at doses100-fold greater than its ED₅₀.

[0030] FIGS. 15A-15B show a comparison of CGX-1160, CGX-1063 and NT onchange in body temperature. FIG. 15A shows time to peak effect andduration of each compound, and FIG. 15B shows dose-response of eachcompound.

[0031] FIGS. 16A-16C show hypothermic dose-effect and duration ofCGX-1160, CGX-1063 and NT. In FIG. 16A, CGX-1160 caused hypothermia onlyat doses 100-500 times greater than ED₅₀. FIG. 16B shows thelong-lasting hypothermic effect of CGX-1063 at doses 10-fold higher thanED₅₀.(100 pmol). In FIG. 16C, NT had a hypothalamic effect at doses10-100 times higher than its ED₅₀.

[0032]FIG. 17 shows effects of Thr₁₀-g Contulakin-G (CGX-1160; 100 pmoli.c.v.) on D-amphetamine-stimulated locomotor activity as measured bydistance traveled. Abbreviations: sal-sal: i.p. treatment was saline,i.c.v. treatment was saline; amphet (3 mg/kg)-sal: i.p. treatment wasD-amphetamine sulphate (3 mg/kg), i.c.v. treatment was saline; amphet(10 mg/kg)-sal: i.p. treatment was D-amphetamine sulphate (10 mg/kg),i.c.v. treatment was saline; sal-ctl: i.p. treatment was saline, i.c.v.treatment was Thr₁₀-g contulakin-G (100 pmol); amphet (3 mg/kg)-ctl:i.p. treatment was D-amphetamine sulphate (3 mg/kg), i.c.v. treatmentwas Thr₁₀-g contulakin-G (100 pmol). Each bar shows the mean±SEM of 3-7mice per group. a: P<0.05 vs saline-saline treated group (sal-sal); b:P<0.05 vs D-amphetamine-saline group (amphet (3 mg/kg)-sal).

[0033]FIG. 18 shows the effects of Thr₁₀-g Contulakin-G (CGX-1160; 100pmol i.c.v.) on D-amphetamine-stimulated locomotor activity as measuredby time spent ambulatory (s). Abbreviations: sal-sal: i.p. treatment wassaline, i.c.v. treatment was saline; amphet (3 mg/kg)-sal: i.p.treatment was D-amphetamine sulphate (3 mg/kg), i.c.v. treatment wassaline; amphet (10 mg/kg)-sal: i.p. treatment was D-amphetamine sulphate(10 mg/kg), i.c.v. treatment was saline; sal-ctl: i.p. treatment wassaline, i.c.v. treatment was Thr ₁₀-g contulakin-G (100 pmol); amphet (3mg/kg)-ctl: i.p. treatment was D-amphetamine sulphate (3 mg/kg), i.c.v.treatment was Thr₁₀-g contulakin-G (100 pmol). Each bar shows themean±SEM of 3-7 mice per group. a: P<0.05 vs saline-saline treated group(sal-sal); b: P<0.05 vs D-amphetamine-saline group (amphet (3mg/kg)-sal).

[0034]FIG. 19 shows CGX-1160 and CGX-1063 dose-dependently protectagainst audiogenic seizures following i.c.v. administration in Fringsmice, at doses well below minimal motor impairing doses. Each pointrepresents the percent protection (toxic in groups of at least fourmice).

[0035]FIG. 20 shows CGX-1160's long-lasting efficacy in blockingaudiogenic seizures following i.c.v. administration in Frings mice.Neurotensin is only 50% effective following i.c.v. administration of upto 5 nmol. Each point represents the percent protection in a group offour mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present invention is directed to contulakin-G (which is thenative glycosylated peptide), a des-glycosylated contulakin-G (termedThr₁₀-contulakin-G), and derivatives thereof, to a cDNA clone encoding aprecursor of this mature peptide and to a precursor peptide. Theinvention is further directed to the use of this peptide as atherapeutic for anti-seizure, anti-inflammatory, anti-shock,anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson'sdisease, gastrointestinal disorders, depressive states, cognitivedysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack,mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette'ssyndrome, Huntington's chorea, vascular leakage, anti-arteriosclerosis,vascular and vasodilation disorders, as well as neurological,neuropharmalogical and neuropsychopharmacological disorders.

[0037] The present invention is directed to contulakin-G andcontulakin-G analogues as described above. These peptides may containsingle or multiple glycan post-translational modifications at one ormore, up to all, of the hydroxyl sites of the peptides. The glycans areas described herein. The native O-glycan attached to contulakin-G isshown in FIG. 2. FIG. 3 shows analogs of the glycan which can beattached to one or more residues of contulakin-G. In this figure, R₁ isan amino capable of being derivatized with a gylcan either chemically orenzymatically; R₂ is OH, NH₂, NHSO₃Na, NHAc, O-sulphate, O-phosphate, orO-glycan; R₃ is H, SO₃, PO₃, acetyl, sialic acid or monosaccharide; R₄is H, SO₃, PO₃, acetyl or monosaccharide; R₅ is OH, NH₂, NHSO₃Na, NHAc,O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl; R₆ is H, SO₃,PO₃, acetyl or monosaccharide; R₇ is H, SO₃, PO₃, acetyl ormonosaccharide; R₈ is H, SO₃, PO₃, acetyl or monosaccharide; n is 0-4and m is 1-4.

[0038] The preferred core glycans which can be used to modifycontulakin-G or analogs disclosed herein are shown in FIG. 4. Furtherbranching from these cores using the monosaccharides described hereinmay also be made. Preferred glycosidic linkages are specified by cores 5and 7 of FIG. 4 with further homolgation of the glycan at positions 3, 4and 6 of the GalNAc template using the monosaccharides described hereinAny free hydroxy function may be O-sulphated, O-phosphorylated orO-aceylated.

[0039] The glycosylated conopeptide (contulakin-G or CGX-1160) hashigher in vivo potency than the unglycosylated conopeptide(Thr₁₀-contulakin-G or CGX-1063), although their in vitro potencies areabout the same. The glycosylation may be important for better bindingwith the receptor, and/or enhanced delivery of the conopeptide to itssite of action, and/or inhibition of degradation of the conopeptide.

[0040] The present invention is further directed to DNA sequence codingfor contulakin-G as described in further herein. The invention isfurther directed to the propeptide for contulakin-G as described infurther detail herein.

[0041] The present invention relates to a novel linear glycosylatedcontulakin-G, and derivatives thereof that are useful as pharmaceuticalagents, to methods for their production, to pharmaceutical compositionswhich include these compounds and a pharmaceutically acceptable carrier,and to pharmaceutical methods of treatment. The novel compounds of thepresent invention are central nervous system agents and their biologicalactions are effected at a novel “Contulakin-G binding site on theneurotensin receptor”. More particularly, the novel compounds of thepresent invention are analgesics, anti-inflammatory agents,antipsychotic agents for treating psychoses such as schizophrenia anddisplay potent anti-seizure properties in established animal models ofepilepsy.

[0042] PAIN: Chronic or intractable pain, such as may occur inconditions such as bone degenerative diseases and cancer, is adebilitating condition which is treated with a variety of analgesicagents, and often opioid compounds, such as morphine.

[0043] In general, brain pathways governing the perception of pain arestill incompletely understood, sensory afferent synaptic connections tothe spinal cord, termed “nociceptive pathways” have been documented insome detail. In the first leg of such pathways, C- and A-fibers whichproject from peripheral sites to the spinal cord carry nociceptivesignals. Polysynaptic junctions in the dorsal horn of the spinal cordare involved in the relay and modulation of sensations of pain tovarious regions of the brain, including the periaqueductal grey region.Analgesia, or the reduction of pain perception, can be effected directlyby decreasing transmission along such nociceptive pathways. Analgesicopiates are thought to act by mimicking the effects of endorphin orenkephalin peptide-containing neurons, which synapse presynaptically atthe C- or A-fiber terminal and which, when they fire, inhibit release ofneurotransmitters, including substance P. Descending pathways from thebrain are also inhibitory on C- and A-fiber firing.

[0044] Certain types of pain have complex etiologies. For example,neuropathic pain is generally a chronic condition attributable to injuryor partial transection of a peripheral nerve. This type of pain ischaracterized by hyperesthesia, or enhanced sensitivity to externalnoxious stimuli. The hyperesthetic component of neuropathic pain doesnot respond to the same pharmaceutical interventions as does moregeneralized and acute forms of pain.

[0045] Opioid compounds such as morphine, while effective in producinganalgesia for many types of pain, are not always effective, and mayinduce tolerance in patients. When a subject is tolerant to opioidnarcotics, increased doses are required to achieve a satisfactoryanalgesic effect. These compounds can produce side effects, such asrespiratory depression, which can be life threatening. In addition,opioids frequently produce physical dependence in patients. Dependenceappears to be related to the dose of opioid taken and the period of timeover which it is taken by the subject. For this reason, alternatetherapies for the management of chronic pain are widely sought after. Inaddition, compounds which serve as either a replacement for or as anadjunct to opioid treatment in order to decrease the dosage of analgesiccompound required, have utility in the treatment of pain, particularlypain of the chronic, intractable type.

[0046] Since contulakin-G has been shown to act at a site on certainneurotensin receptors, and neurotensin has been shown to have analgesicactions (Clineschmidt et al. 1979), then contulakin-G like conopeptidesare useful for the treatment of pain and related disorders.

[0047] SCHIZOPHRENIA: Schizophrenia is a neurogenic disorder that iscurrently treated primarily with neuroleptic compounds such asphenothiazines and butyrophenones, which block dopamine receptors. Sincecontulakin-G has been shown to act at a site on certain neurotensinreceptors, and neurotensin actions are implicated in the etiology ofschizophrenia (Nemeroff et al. 1992), then contulakin-G likeconopeptides are useful for the treatment of schizophrenia and relateddisorders.

[0048] The in vitro selection criteria for conopeptides useful intreating schizophrenia, include: a) activation of Contulakin-G sites; b)high affinity reversible binding to a Contulakin-G binding sitelocalized to the limbic region of the brain, and c) inhibition ofdopamine release from brain regions, particularly limbic brain regions.

[0049] Compounds exhibiting sufficiently high activities in the above invitro screening assays are then tested in an animal model used inscreening anti-psychotic compounds.

[0050] TARDIVE DYSKINESIA AND OTHER ACUTE DYSTONIC REACTIONS: Tardivedyskinesia and acute dystonic reactions are movement disorders that arecommonly produced as side effects of anti-psychotic therapy employingdopamine antagonists, such as haloperidol. These disorders arecharacterized by supersensitivity of dopamine receptors in certainregions of the brain associated with control of movement, particularlythe basal ganglia. Currently, intermittent antipsychotic therapy is usedin attempt to avoid onset of the disorder, and such disorders aretreated by withdrawal of therapy.

[0051] Criteria for selection of a conopeptide for treatment of tardivedyskinesia include: a) activation of Contulakin-G sites; b) highaffinity reversible binding to the Contulakin-G site; c) inhibition ofdopamine release from striatal brain regions, and other regions of thebasal ganglia, and d) a ratio of inhibition of dopamine release in thebasal ganglia to inhibition of dopamine release in the limbic regions.

[0052] Compounds showing sufficiently high activities in in vitroscreening assays are then tested in the rat striatal turning model,described above. Compounds useful in the method of treating suchmovement disorders, when injected to the striatum on the side of thebrain contralateral to the lesion, correct the turning behavior.

[0053] INFLAMMATION: A neurogenic component of inflammation has beendescribed, in that blockade of the sympathetic nervous system, andparticularly blockade of beta-adrenergic receptors, is helpful inreducing inflammatory joint damage. Compounds useful in the treatment ofinflammation would be expected to have the following in vitroproperties: a) activation of novel Contulakin-G sites; b) high affinitybinding to the Contulakin-G binding sites, and c) inhibition ofnorepinephrine release from nervous tissue. Compounds exhibitingsufficiently high activities in such in vitro screening assays aretested in an animal model of rheumatoid arthritis.

[0054] EPILEPSY: Epilepsy is a general term which describes disorders ofthe central nervous system characterized by repeated episodes ofseizures. Such seizures may involve the sensory, autonomic or motornervous systems and are recognized electrophysiologically by thepresence of abnormal electric discharges in the brain. Thepathophysiology of such abnormal discharge activity is not wellunderstood; however, there is evidence that loss of inhibitory neuralinput, such as GABA input, is involved in at least some epilepticseizures.

[0055] The ability of certain of the benzodiazepines (e.g., diazepam) torepress or inhibit epileptic episodes is considered by some to beevidence of a GABAergic pathophysiology in seizure activity, since thesedrugs are known to potentiate GABAergic neural inhibition via an effecton the GABA receptor-associated chloride ion channel. Biochemicaleffects of other anti-epileptic compounds include stabilization ofexcitable membranes by inhibition of voltage-sensitive sodium orpotassium channels (phenytoin), and general depression of neuronalfunction characterized by facilitation of GABAergic transmission,inhibition of the effects of excitatory (glutaminergic)neurotransmission and depression of neurotransmitter release(phenobarbital).

[0056] Compounds useful in the treatment of epilepsy would be expectedto have the following in vitro properties: a) activation of novelContulakin-G sites; b) high affinity binding to the contulakin-Gconopeptide binding sites, and c) inhibition of excitatoryneurotransmitter release from nervous tissue. Compounds exhibitingsufficiently high activities in such in vitro screening assays faretested in an established animal model of epilepsy.

[0057] In addition to the above specific disorders, since the peptides,derivatives and analogs of the present invention have been found to bindto the neurotensin receptor, these compounds are also useful inconnection with conditions associated with the neurotensin receptor andfor which neurotensin-like compounds or other compounds have been shownto be active. These activities include: methamphetamine antagonists,antipsychotic agents, cerebral medicaments, analgesic agents,anti-endotoxin shock effect, protease inhibition action (ananti-thrombin action, an anti-plasmin action), a hypotensive action, ananti-DIC action, an anti-allergic action, a wound healing action,cerebral edema, an edema of the lung, an edema of the trachea, athrombus, an arteriosclerosis, a burn, and a hypertension, allergicdiseases (such as a bronchial asthma and a pollenosis), reducinghemorrhage from a sharp trauma such as an injured tissue portion at thetime of surgical operation, a lacerated wound of a brain or othertissues caused by a traffic accident and the like, and for relaxing andcuring swelling, pain inflammation caused by trauma, suppressinginternal hemorrhage caused by a dull trauma, edemata and inflammationwhich are accompanied with the internal hemorrhage, suppression andimprovement of cerebral edemata by suppressing a leakage of bloodcomponents to a tissue matrix found in cerebral ischemetic diseaseswhich include cerebral infarctions (e.g., a cerebral thrombus and acerebral embolism), intracranial hemorrhages (e.g., a cerebralhemorrhage and a subarachnoidal hemorrhage), a transient cerebralischemic attack, acute cerebral blood vessel disorders in a hypertensiveencephalopathy, suppression and improvement of burns, chilblains, otherskin inflammations and swelling, an upper tracheal inflammation, anasthma, nasal congestion, a pulmonary edema, and inflammable disorderscaused by endogenous and exogenous factors, which directly damagevascular endothelia and mucous membranes, such as an environmentalchemical substance, chemotherapeutics of cancer, an endotoxin, and aninflammation mediator.

[0058] The conopeptides of the present invention are identified byisolation from Conus venom. Alternatively, the conopeptides of thepresent invention are identified using recombinant DNA techniques byscreening cDNA libraries of various Conus species using conventionaltechniques with degenerate probes. Clones which hybridize to theseprobes are analyzed to identify those which meet minimal sizerequirements, i.e., clones having approximately 300 nucleotides (for apropeptide), as determined using PCR primers which flank the cDNAcloning sites for the specific cDNA library being examined. Theseminimal-sized clones are then sequenced. The sequences are then examinedfor the presence of a peptide having the characteristics noted above forconopeptides. The biological activity of the peptides identified by thismethod is tested as described herein, in U.S. Pat. No. 5,635,347 orconventionally in the art.

[0059] These peptides are sufficiently small to be chemicallysynthesized. General chemical syntheses for preparing the foregoingconopeptides are described hereinafter, along with specific chemicalsynthesis of conopeptides and indications of biological activities ofthese synthetic products. Various ones of these conopeptides can also beobtained by isolation and purification from specific Conus species usingthe techniques described in U.S. Pat. No. 4,447,356 (Olivera et al.,1984), U.S. Pat. No. 5,514,774 (Olivera et al., 1996) and U.S. Pat. No.5,591,821 (Olivera et al., 1997), the disclosures of which areincorporated herein by reference.

[0060] Although the conopeptides of the present invention can beobtained by purification from cone snails, because the amounts ofconopeptides obtainable from individual snails are very small, thedesired substantially pure conopeptides are best practically obtained incommercially valuable amounts by chemical synthesis using solid-phasestrategy. For example, the yield from a single cone snail may be about10 micrograms or less of conopeptide. By “substantially pure” is meantthat the peptide is present in the substantial absence of otherbiological molecules of the same type; it is preferably present in anamount of at least about 85% purity and preferably at least about 95%purity. Chemical synthesis of biologically active conopeptides dependsof course upon correct determination of the amino acid sequence. Thus,the conopeptides of the present invention may be isolated, synthesizedand/or substantially pure.

[0061] The conopeptides can also be produced by recombinant DNAtechniques well known in the art. Such techniques are described bySambrook et al. (1989). The peptides produced in this manner areisolated, reduced if necessary, and oxidized to form the correctdisulfide bonds, if present in the final molecule.

[0062] One method of forming disulfide bonds in the conopeptides of thepresent invention is the air oxidation of the linear peptides forprolonged periods under cold room temperatures or at room temperature.This procedure results in the creation of a substantial amount of thebioactive, disulfide-linked peptides. The oxidized peptides arefractionated using reverse-phase high performance liquid chromatography(HPLC) or the like, to separate peptides having different linkedconfigurations. Thereafter, either by comparing these fractions with theelution of the native material or by using a simple assay, theparticular fraction having the correct linkage for maximum biologicalpotency is easily determined. It is also found that the linear peptide,or the oxidized product having more than one fraction, can sometimes beused for in vivo administration because the cross-linking and/orrearrangement which occurs in vivo has been found to create thebiologically potent conopeptide molecule. However, because of thedilution resulting from the presence of other fractions of lessbiopotency, a somewhat higher dosage may be required.

[0063] The peptides are synthesized by a suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation or by classical solution couplings.

[0064] In conventional solution phase peptide synthesis, the peptidechain can be prepared by a series of coupling reactions in whichconstituent amino acids are added to the growing peptide chain in thedesired sequence. Use of various coupling reagents, e.g.,dicyclohexylcarbodiimide or diisopropyl-carbonyldimidazole, variousactive esters, e.g., esters of N-hydroxyphthalimide orNhydroxy-succinimide, and the various cleavage reagents, to carry outreaction in solution, with subsequent isolation and purification ofintermediates, is well known classical peptide methodology. Classicalsolution synthesis is described in detail in the treatise, “Methoden derOrganischen Chemie (Houben-Weyl): Synthese von Peptiden,” (1974).Techniques of exclusively solid-phase synthesis are set forth in thetextbook, “Solid-Phase Peptide Synthesis,” (Stewart and Young, 1969),and are exemplified by the disclosure of U.S. Pat. No. 4,105,603 (Valeet al., 1978). The fragment condensation method of synthesis isexemplified in U.S. Pat. No. 3,972,859 (1976). Other available synthesesare exemplified by U.S. Pat. No. 3,842,067 (1974) and U.S. Pat. No.3,862,925 (1975). The synthesis of peptides containing γ-carboxyglutamicacid residues is exemplified by Rivier et al. (1987), Nishiuchi et al.(1993) and Zhou et al. (1996). Synthesis of conopeptides have beendescribed in U.S. Pat. No. 4,447,356 (Olivera et al., 1984), U.S. Pat.No. 5,514,774 (Olivera et al., 1996) and U.S. Pat. No. 5,591,821(Olivera et al., 1997).

[0065] Common to such chemical syntheses is the protection of the labileside chain groups of the various amino acid moieties with suitableprotecting groups which will prevent a chemical reaction from occurringat that site until the group is ultimately removed. Usually also commonis the protection of an α-amino group on an amino acid or a fragmentwhile that entity reacts at the carboxyl group, followed by theselective removal of the α-amino protecting group to allow subsequentreaction to take place at that location. Accordingly, it is common that,as a step in such a synthesis, an intermediate compound is producedwhich includes each of the amino acid residues located in its desiredsequence in the peptide chain with appropriate side-chain protectinggroups linked to various ones of the residues having labile side chains.

[0066] As far as the selection of a side chain amino protecting group isconcerned, generally one is chosen which is not removed duringdeprotection of the α-amino groups during the synthesis. However, forsome amino acids, e.g., His, protection is not generally necessary. Inselecting a particular side chain protecting group to be used in thesynthesis of the peptides, the following general rules are followed: (a)the protecting group preferably retains its protecting properties and isnot split off under coupling conditions, (b) the protecting group shouldbe stable under the reaction conditions selected for removing theα-amino protecting group at each step of the synthesis, and (c) the sidechain protecting group must be removable, upon the completion of thesynthesis containing the desired amino acid sequence, under reactionconditions that will not undesirably alter the peptide chain.

[0067] It should be possible to prepare many, or even all, of thesepeptides using recombinant DNA technology. However, when peptides arenot so prepared, they are preferably prepared using the Merrifieldsolid-phase synthesis, although other equivalent chemical synthesesknown in the art can also be used as previously mentioned. Solid-phasesynthesis is commenced from the C-terminus of the peptide by coupling aprotected α-amino acid to a suitable resin. Such a starting material canbe prepared by attaching an α-amino-protected amino acid by an esterlinkage to a chloromethylated resin or a hydroxymethyl resin, or by anamide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine(MBHA) resin. Preparation of the hydroxymethyl resin is described byBodansky et al. (1966). Chloromethylated resins are commerciallyavailable from Bio Rad Laboratories (Richmond, Calif.) and from Lab.Systems, Inc. The preparation of such a resin is described by Stewartand Young (1969). BHA and MBHA resin supports are commerciallyavailable, and are generally used when the desired polypeptide beingsynthesized has an unsubstituted amide at the C-terminus. Thus, solidresin supports may be any of those known in the art, such as one havingthe formulae —O—CH₂-resin support, —NH BHA resin support, or —NH—MBHAresin support. When the unsubstituted amide is desired, use of a BHA orMBHA resin is preferred, because cleavage directly gives the amide. Incase the N-methyl amide is desired, it can be generated from an N-methylBHA resin. Should other substituted amides be desired, the teaching ofU.S. Pat. No. 4,569,967 (Kornreich et al., 1986) can be used, or shouldstill other groups than the free acid be desired at the C-terminus, itmay be preferable to synthesize the peptide using classical methods asset forth in the Houben-Weyl text (1974).

[0068] The C-terminal amino acid, protected by Boc or Fmoc and by aside-chain protecting group, if appropriate, can be first coupled to achloromethylated resin according to the procedure set forth in Horiki etal. (1978), using KF in DMF at about 60° C. for 24 hours with stirring,when a peptide having free acid at the C-terminus is to be synthesized.Following the coupling of the BOC-protected amino acid to the resinsupport, the α-amino protecting group is removed, as by usingtrifluoroacetic acid (TFA) in methylene chloride or TFA alone. Thedeprotection is carried out at a temperature between about 0° C. androom temperature. Other standard cleaving reagents, such as HCl indioxane, and conditions for removal of specific α-amino protectinggroups may be used as described in Schroder and Lubke (1965).

[0069] After removal of the α-amino-protecting group, the remainingα-amino- and side chain-protected amino acids are coupled step-wise inthe desired order to obtain the intermediate compound definedhereinbefore, or as an alternative to adding each amino acid separatelyin the synthesis, some of them may be coupled to one another prior toaddition to the solid phase reactor. Selection of an appropriatecoupling reagent is within the skill of the art. Particularly suitableas a coupling reagent is N,N′-dicyclohexylcarbodiimide (DCC, DIC, HBTU,HATU, TBTU in the presence of HoBt or HoAt).

[0070] The activating reagents used in the solid phase synthesis of thepeptides are well known in the peptide art. Examples of suitableactivating reagents are carbodiimides, such asN,N′-diisopropylcarbodiimide andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. Other activatingreagents and their use in peptide coupling are described by Schroder andLubke (1965) and Kapoor (1970).

[0071] Each protected amino acid or amino acid sequence is introducedinto the solid-phase reactor in about a twofold or more excess, and thecoupling may be carried out in a medium of dimethylformamide(DMF):CH₂Cl₂ (1:1) or in DMF or CH₂Cl₂ alone. In cases whereintermediate coupling occurs, the coupling procedure is repeated beforeremoval of the α-amino protecting group prior to the coupling of thenext amino acid. The success of the coupling reaction at each stage ofthe synthesis, if performed manually, is preferably monitored by theninhydrin reaction, as described by Kaiser et al. (1970). Couplingreactions can be performed automatically, as on a Beckman 990 automaticsynthesizer, using a program such as that reported in Rivier et al.(1978).

[0072] After the desired amino acid sequence has been completed, theintermediate peptide can be removed from the resin support by treatmentwith a reagent, such as liquid hydrogen fluoride or TFA (if using Fmocchemistry), which not only cleaves the peptide from the resin but alsocleaves all remaining side chain protecting groups and also the α-aminoprotecting group at the N-terminus if it was not previously removed toobtain the peptide in the form of the free acid. If Met is present inthe sequence, the Boc protecting group is preferably first removed usingtrifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptidefrom the resin with HF to eliminate potential S-alkylation. When usinghydrogen fluoride or TFA for cleaving, one or more scavengers such asanisole, cresol, dimethyl sulfide and methylethyl sulfide are includedin the reaction vessel.

[0073] Cyclization of the linear peptide is preferably affected, asopposed to cyclizing the peptide while a part of the peptido-resin, tocreate bonds between Cys residues. To effect such a disulfide cyclizinglinkage, fully protected peptide can be cleaved from a hydroxymethylatedresin or a chloromethylated resin support by ammonolysis, as is wellknown in the art, to yield the fully protected amide intermediate, whichis thereafter suitably cyclized and deprotected. Alternatively,deprotection, as well as cleavage of the peptide from the above resinsor a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), cantake place at 0° C. with hydrofluoric acid (HF) or TFA, followed byoxidation as described above. A suitable method for cyclization is themethod described by Cartier et al. (1996).

[0074] Muteins, analogs or active fragments, of the foregoingcontulakin-G or Thr₁₀-g contulakin-G are also contemplated here. See,e.g., Hammerland et al (1992). Derivative muteins, analogs or activefragments of the conotoxin peptides may be synthesized according toknown techniques, including conservative amino acid substitutions, suchas outlined in U.S. Pat. No. 5,545,723 (see particularly col. 2, line 50to col. 3, line 8); U.S. Pat. No. 5,534,615 (see particularly col. 19,line 45 to col. 22, line 33); and U.S. Pat. No. 5,364,769 (seeparticularly col. 4, line 55 to col. 7, line 26), each incorporatedherein by reference.

[0075] Pharmaceutical compositions containing a compound of the presentinvention as the active ingredient can be prepared according toconventional pharmaceutical compounding techniques. See, for example,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa.). Typically, an antagonistic amount of the activeingredient will be admixed with a pharmaceutically acceptable carrier.The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., intravenous, oral orparenteral.

[0076] For oral administration, the compounds can be formulated intosolid or liquid preparations such as capsules, pills, tablets, lozenges,melts, powders, suspensions or emulsions. In preparing the compositionsin oral dosage form, any of the usual pharmaceutical media may beemployed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents, suspending agents, andthe like in the case of oral liquid preparations (such as, for example,suspensions, elixirs and solutions); or carriers such as starches,sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like in the case of oral solidpreparations (such as, for example, powders, capsules and tablets).Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe sugar-coated or enteric-coated by standard techniques.

[0077] For parenteral administration, the compound may be dissolved in apharmaceutical carrier and administered as either a solution or asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they may also be dissolved in cerebrospinal fluid.

[0078] Administration of the active agent according to this inventionmay be achieved using any suitable delivery means, including:

[0079] (a) pump (see, e.g., Annals of Pharmacotherapy, 27:912 (1993);Cancer, 41:1270 (1993); Cancer Research, 44:1698 (1984));

[0080] (b), microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883;4,353,888; and 5,084,350);

[0081] (c) continuous release polymer implants (see, e.g., U.S. Pat. No.4,883,666);

[0082] (d) macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761,5,158,881, 4,976,859 and 4,968,733 and published PCT patent applicationsWO92/19195, WO 95/05452);

[0083] (e) naked or unencapsulated cell grafts to the CNS (see, e.g.,U.S. Pat. Nos. 5,082,670 and 5,618,531);

[0084] (f) injection, either subcutaneously, intravenously,intra-arterially, intramuscularly, or to other suitable site; or

[0085] (g) oral administration, in capsule, liquid, tablet, pill, orprolonged release formulation.

[0086] In one embodiment of this invention, an active agent is delivereddirectly into the CNS, preferably to the brain ventricles, brainparenchyma, the intrathecal space or other suitable CNS location, mostpreferably intrathecally.

[0087] Alternatively, targeting therapies may be used to deliver theactive agent more specifically to certain types of cells, by the use oftargeting systems such as antibodies or cell-specific ligands. Targetingmay be desirable for a variety of reasons, e.g. if the agent isunacceptably toxic, if it would otherwise require too high a dosage, orif it would not otherwise be able to enter target cells.

[0088] The active agents, which are peptides, can also be administeredin a cell based delivery system in which a DNA sequence encoding anactive agent is introduced into cells designed for implantation in thebody of the patient, especially in the spinal cord region. Suitabledelivery systems are described in U.S. Pat. No. 5,550,050 and publishedPCT Application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452,WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635.Suitable DNA sequences can be prepared synthetically for each activeagent on the basis of the developed sequences and the known geneticcode.

[0089] The active agent is preferably administered in a therapeuticallyeffective amount. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofthe condition being treated. Prescription of treatment, e.g. decisionson dosage, timing, etc., is within the responsibility of generalpractitioners or specialists, and typically takes into account thedisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners. Examples of techniques and protocols can be found inRemington's Pharmaceutical Sciences. Typically, the active agents of thepresent invention exhibit their effect at a dosage range of from about0.001 μg/kg to about 500 μg/kg, preferably from about 0.01 μg/kg toabout 100 μg/kg, of the active ingredient, more preferably, from about0.10 μg/kg to about 50 μg/kg, and most preferably, from about 1 μg/kg toabout 10 μg/kg. A suitable dose can be administered in multiplesub-doses per day. Typically, a dose or sub-dose may contain from about0.1 μg to about 500 μg of the active ingredient per unit dosage form. Amore preferred dosage will contain from about 0.5 μg to about 100 μg ofactive ingredient per unit dosage form. Dosages are generally initiatedat lower levels and increased until desired effects are achieved.

EXAMPLES

[0090] The present invention is further detailed in the followingexamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below are utilized. Theabbreviations used are: Bop, benzotriazoyloxy-tris (dimethyl amino)phosphonium hexafluorophosphate; Boc, tert butyloxycarbonyl; Fmoc,9-fluoroenylmethoxy carbonyl; Gal, galactose; GalNAc, N-acetylgalactosamine; hNTR1, human neurotensin type 1 receptor; Hex, hexose;HexNAc, N-acetyl hexosamine; icv, intra cerebroventricular; LSI, liquidsecondary ionization; MALD, matrix assisted laser desorption; MS, massspectrometry; mNTR3, mouse neurotensin type 3 receptor; nano-ESI,nano-electrospray; NMP, N-methylpyrrolidone; NMR, nuclear magneticresonance; ppm, parts per million; rNTR1, rat neurotensin type 1receptor; rNTR2, rat neurotensin type 2 receptor; RP-HPLC, reversephase-high performance liquid chromatography. Amino acids are indicatedby the standard three or one letter abbreviations.

Example 1 Experimental Procedures for Initial Analysis of Contulakin-G

[0091] 1. Crude venom. Conus geographus specimens were collected fromMarinduque Is. in the Philippines. The crude venom was obtained bydissection of the venom duct gland and then freeze dried and stored at−70° C.

[0092] 2. Peptide purification. Freeze dried C. geographus venom (1 g)was extracted with 1.1% acetic acid and chromatographed on a SephadexG-25 column eluted with 1.1% acetic acid as previously described(Olivera et al., 1984). A peptide that makes mice sluggish andunresponsive was purified by a series of RP-HPLC purifications onpreparative and semi-preparative and analytical reverse phase C₁₈columns. A gradient of acetonitrile in 0.1% trifluoroacetic acid wasused to elute the peptide from the columns. The major species wasre-purified prior to further characterization. Briefly, one gram ofcrude lyophilized venom from Conus geographus was extracted and appliedon a Sephadex G-25 column as previously described (Olivera et al.,1984). Three successive fractions containing paralytic and sleeperactivities (Ve/Vo=1.37 to 1.41) were pooled, applied on a preparativereversed phase Vydac C₁₈ column and eluted with a gradient ofacetonitrile in 0.1% trifluroacetic acid (FIG. 1). The componentindicated by an arrow in FIG. 1 caused wobbling and death whenadministered icv in mice. This was applied on a semipreparative C₁₈column, eluted with 12-42% acetonitrile gradient in 0.1% trifluroaceticacid. The component which made mice unresponsive when administered icv,was further purified with an isocratic elution at 20.4% acetonitrile in0.1% trifluroacetic acid. A mouse injected icv with an aliquot of thecomponent had trouble righting itself in 5 min and became very sluggishwithin 12 min. In approximately 25-30 min, the mouse was stretched outand laid on its stomach.

[0093] 3. Bioactivity. Typically, mice injected icv with the partiallypurified native peptide initially had trouble righting after 5 min,became sluggish after 12 min and then rested on their stomachs after 30min. These signs were used as an assay to identify the biologicallyactive peptide during purification.

[0094] 4. Enzyme hydrolysis. Approximately 180 pmol of the peptide (6μL) was incubated with 7 mU β-Galactosidase (bovine testes) (2 μL) in 50of μL 50 mM citrate/phosphate buffer (pH 4.5) for 53 hr at 32° C.Approximately 60 pmol of the peptide (2 μL) was incubated with 2 mUO-glycosidase (Diplococcus pneumoniae) (2 μL) in 50 μL of 20 mMcacodylic acid (pH 6.0) for 19 hr at 32° C.

[0095] 5. Chemical sequence and amino acid analysis. Automated chemicalsequence analysis was performed on a 477A Protein Sequencer (AppliedBiosystems, Foster City, Calif.). Amino acid analysis was carried outusing pre-column derivatization. Approximately 500 pmol of thecontulakin-G was sealed under vacuum with concentrated HCl, hydrolyzedat 110° C. for 24 hr, lyophilized and then derivatized witho-phthalaldehyde. The derivatized amino acids were then analyzed withRP-HPLC.

[0096] 6. Mass spectrometry. Matrix assisted laser desorption (MALD)(Hillenkamp et al., 1993) mass spectra were measured using a ‘BrukerREFLEX’ (Bruker Daltonics, Billerica, Mass.) time-of-flight (Cotter,1989) mass spectrometer fitted with a gridless reflectron, an N₂ laserand a 100 MHz digitizer. An accelerating voltage of +31 kV and areflector voltage between 1.16 and 30 kV were employed for the postsource decay (Spengler et al., 1992) measurements. The sample (in 0.1%aqueous trifluoroacetic acid) was applied with α-cyano-4-hydroxycinnamicacid. Liquid secondary ionization (LSI) (Barber et al., 1982) massspectra were measured using a Jeol HX110 (Jeol, Tokyo, Japan) doublefocusing mass spectrometer operated at 10 kV accelerating voltage, 1000or 3000 resolution. The sample (in 0.1% aqueous trifluoroacetic acid and25% acetonitrile) was mixed in a thioglycerol and dithiothreitol matrix.Nano-electrospray (nano-ESI) mass spectra were measured using an Esquireion trap mass spectrometer (Bruker Daltonics, Billerica, Mass.). TheRP-HPLC purified sample, collected in 0.1% aqueous trifluoro-acetic acidand acetonitrile was diluted in methanol 1% acetic acid, transferred toa nanospray capillary and analyzed. The mass accuracy was typicallybetter than 1000 ppm for the time-of-flight instrument, 200 ppm for theion trap instrument and 20-100 ppm for the double focusing massspectrometer depending on the resolving power settings of the magneticsector instrument employed.

[0097] 7. Synthesis of contulakin-G. The solid-phase glycopeptidesynthesis was carried out manually using Fmoc chemistry, with t-butylether side chain protection for tyrosine and serine, N-t-Boc side chainprotection for lysine, and t-butyl ester side chain protection forglutamic acid (protected amino acids were obtained from Bachem,Torrance, Calif.). Starting with a Wang resin, the amino acids werecoupled withBop/diisopropylethylamine/N-methylpyrrolidone/dichloromethane (Stewartet al., 1984; LeNguyen et al., 1986) and the N-deprotections were donewith N-methylpyrrolidone/piperidine (Stewart et al., 1984; LeNguyen etal., 1986). The Wang resin was prepared at The Salk Institute with asubstitution of 0.2 nmol/g. After coupling of the first six amino acids,the resin was coupled with peracetylatedFmoc-Oβ-D-Galp-(1→3)-α-D-GalpNAc-(1→O)-threonine, synthesized asdescribed elsewhere (Luning et al., 1989), followed by single couplingof the remaining nine amino acids in the sequence. Care was taken toremove acetic acid and acetate impurities from the glycosylated aminoacids; this included chromatographic purification on silica gel usingdichloromethane-ethyl acetate 4:1 as eluant, concentration and finallyophilization of the product from benzene. Non-glycosylated peptide wassimilarly synthesized using Fmoc-threonine (Bachem, Torrance, Calif.).The resin was subjected to cleavage conditions (95% trifluoroaceticacid/5% anisole (Stewart et al., 1984)), and in the case of theglycopeptide, the resulting peracetylated glycopeptide was isolated withRP-HPLC, the major component m/z 2322.3 (MALD analysis) corresponding tothe desired product (2322.0 Da). After lyophilization, the peracetylatedglycopeptide was treated with 20 μL of sodium methoxide (Sigma, StLouis, Mo.) (50 mM) in dry methanol for 1 minute (to remove O-acetylgroups on the sugar (Norberg et al., 1994)) and lyophilized at −20° C.The deacetylated sample was loaded onto a Waters Prep LC/System 500Aequipped with gradient controller, Waters Model 450 Variable WavelengthDetector and Waters 1000 PrepPack cartridge chamber column (65.5×320 mm)packed with Vydac C₁₈ 15-20 μm particles. Flow conditions: wavelength230 nm, AUFS 2.0, flow 100 L/mmin., gradient 20-60% B/60 min; (where theA buffer was 0.1% trifluoroacetic acid in water and the B buffer was0.1% trifluoroacetic acid in 60% aqueous acetonitrile). The fractions(200 mL) were collected manually. The major component, m/z 2069.9 (LSIanalysis), corresponded to the desired product (2069.98 Da). Afterpreparative RP-HPLC purification, sufficient purified contulakin-G wasobtained for analytical characterization and biological studies. A moreextensive characterization of the synthetic contulakin-G including ¹HNMR data will be presented elsewhere.

[0098] 8. Co-elution. The native and synthetic contulakin-G wereanalyzed separately and co-eluted with RP-HPLC, using a 2.1×150 mm VydacC₁₈ column and a 0.5%/min gradient from 0% B to 40% B (where the Abuffer was 0.55% trifluoroacetic acid in water and the B buffer was0.05% trifluoroacetic acid in 90% aqueous acetonitrile).

[0099] 9. Binding studies. The non-glycosylated Thr₁₀-contulakin-G andsynthetic contulakin-G were assayed with the human neurotensin type 1receptor (hNTR1) using a Biomek 1000 robotic workstation for allpipetting steps in the radioligand binding assays, as previouslydescribed (Cusack et al., 1993). Competition binding assays with [³H]neurotensin₁₋₁₃ (1 nM) and varying concentrations of unlabeledneurotensin₁₋₁₃, non-glycosylated Thr₁₀-contulakin-G or syntheticcontulakin-G were carried out with membrane preparations from HEK-293cell line. Nonspecific binding was determined with 11 M unlabeledneurotensin₁₋₁₃ in assay tubes with a total volume of 1 mL. Incubationwas at 20° C. for 30 min. The assay was routinely terminated by additionof cold 0.9% NaCl (5×1.5 mL), followed by rapid filtration through aGF/B filter strip that had been pretreated with 0.2% polyethylenimine.Details of binding assays have been described before (Cusack et al.,1991). The data were analyzed using the LIGAND program (Munson et al.,1980).

[0100] The non-glycosylated Thr₁₀-contulakin-G and syntheticcontulakin-G were separately assayed with the rat neurotensin type 1 andtype 2 receptors (rNTR1 and rNTR2) and mouse neurotensin type 3 receptor(mNTR3). [¹²⁵I-Tyr3] neurotensin₁₋₁₃ was prepared and purified aspreviously described (Saadoul et al., 1984). Stable transfected CHOcells expressing either the rNTR1 (Tanaka et al., 1990) or the rNTR2(cloned in the laboratory of J. Mazella by screening a rat brain cDNAlibrary (Stratagene)) were grown in DMEM containing 10% fetal calf serumand 0.25 mg/mL G418 (Sigma, France). Cell membrane homogenates wereprepared as initially described (Chabry et al., 1994). Proteinconcentration was determined by the Bio-Rad procedure with ovalbumin asthe standard.

[0101] 10. Binding experiments on cell membranes. Membranes (25 μg forNTR2 and 10 μg for NTR1) were incubated with 0.4 nM [¹²⁵I-Tyr³]neurotensin₁₋₁₃ (2000 Ci/mmol) and increasing concentrations ofNeurotensin₁₋₁₃, non-glycosylated Thr₁₀-contulakin-G or syntheticcontulakin-G for 20 min at 25° C. in 250 μl of 50 mM Tris-HCl (pH 7.5)containing 0.1% bovine serum albumin and 0.8 mM 1-10-phenanthroline.Binding experiments were terminated by the addition of 2 mL of ice-coldbuffer followed by filtration through cellulose acetate filters(Sartorius) and washing twice. Radioactivity retained on filters wascounted with a y-counter.

[0102] 11. Binding experiments on solubilized extracts.CHAPS-solubilized extracts (100 μg) were incubated with 0.2 nM[¹²⁵I-Tyr³] neurotensin₁₋ ₁₃ for 1 hr at 0° C. in 250 μL ofTris-glycerol buffer containing 0.1% CHAPS. Bound ligand was separatedfrom free ligand by filtration on GF/B filters pretreated with 0.3%polyethylenimine. Filters were rapidly washed twice with 3 mL of icecold buffer and counted for radioactivity.

[0103] For binding experiments on mNTR3, membrane homogenates from mousebrain were re-suspended in 25 mM Tris-HCl buffer (pH 7.5) containing 10%(w/v) glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin, 1mM iodoacetamide, and 5 mM EDTA (Tris-glycerol buffer). Solubilizationwas carried out by incubating homogenates at a concentration of 10 mg/mLin the Tris-glycerol buffer with 0.625% CHAPS containing 0.125% CHS(Mazella et al., 1988). Solubilized extracts were recovered bycentrifugation at 100,000×g during 30 min at 4° C. and used eitherimmediately or stored at −20° C.

[0104] 12. Phosphoinositides determination. Cells expressing the rNTR1or NTR2 were grown in 12-well plates for 15-18 hr in the presence of 1μCi of myo-[³H]inositol (ICN) in a serum-free HAM's-F-10 medium. Cellswere washed with Earle buffer, pH 7.5, (25 mM Hepes, 25 mM Tris, 140 mMNaCl, 5 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 5 mM glucose) containing0.1% bovine serum albumin, and incubated for 15 min at 37° C. in 900 μlof 30 mM LiCl in Earle buffer. Neurotensin₁₋₁₃ was then added at theindicated concentrations for 15 min. The reaction was stopped by 750 μLof ice cold 10 mM HCOOH, pH 5.5. After 30 min at 4° C., the supernatantwas collected and neutralized by 2.5 mL of 5 mM NH₄OH. Total[³H]phosphoinositides (PIs) were separated from free [³H]inositol onDowex AG-X8 (Bio-Rad) (Van Renterghem et al., 1988) chromatography byeluting successively with 5 mL of water and 4 mL of 40 mM and 1 Mammonium formate, pH 5.5. The radioactivity contained in the 1 Mfraction was counted after addition of 5 mL of Ecolume (ICN).

[0105] 13. Identification of a cDNA clone encoding contulakin-G.Contulakin-G encoding clones were selected from a size-fractionated cDNAlibrary constructed using mRNA obtained from a Conus geographus venomduct as previously described (Colledge et al., 1992). The library wasscreened using a specific probe corresponding to amino acids #10-15 ofthe peptide (5′-ATR ATN GGY TTY TTN GT-3′; SEQ ID NO:3). Theoligonucleotide was end-labeled, hybridized and a secondary screening bypolymerase chain reaction was performed on 10 clones that hybridized tothis probe as previously described (Jimenez et al., 1996). Clonesidentified in the secondary screen were prepared for DNA sequencing aspreviously described (Monje et al., 1993). The nucleic acid sequence wasdetermined according to the standard protocol for Sequenase version 2.0DNA sequencing kit as previously described (Jimenez et al., 1996).

Example 2 Purification of Contulakin-G

[0106] A fraction of Conus geographus venom was detected which made miceexceedingly sluggish. Normally, when mice that are sitting down arepoked with a rod, they immediately get up and run a considerabledistance. Upon i.c.v. injection of the fraction from Conus geographusindicated in FIG. 5, the mice had to be poked with much more forcebefore they got up at all, and after getting up, they would walk one ortwo steps and immediately sit down again. This “sluggish behavior” wasfollowed through several steps of purification, and the apparentlyhomogeneous peptide was further analyzed. This peptide was designatedcontulakin-G (the Filipino woilakin means “has to be pushed or prodded,”from the root word tulak, to push). The “G” indicates that the peptideis from Conus geographus.

Example 3 Biochemical Characterization of the Purified Contulakin-G

[0107] Attempted amino acid sequence analysis of the purified peptiderevealed that the peptide was blocked at the N-terminus. Since mostN-terminally blocked Conus peptides have a pyroglutamate residue atposition 1, the peptide was treated with pyroglutamate aminopeptidase.This resulted in a shift in retention time suggesting removal of apyroglutamate residue. After enzyme treatment, the sequenceSer-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Xaa-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ IDNO:4) was obtained by standard Edman methods confirming removal of thepyroglutamate residue, where Xaa indicates no amino acid was assigned inthe 9th cycle (at position 10) although a very low signal for threoninewas obtained. Amino acid analyses were consistent with the presence ofone threonine residue in the peptide.

[0108] In order to confirm the nature of the amino acid residue inposition 10, a cDNA clone encoding the peptide was isolated. Thenucleotide sequence and presumed amino acid sequence revealed by theclone are shown in Table 1 and in SEQ ID NO:5 and SEQ ID NO:6,respectively. The amino acid sequence of contulakin-G obtained by directEdman sequencing is found encoded towards the C-terminal end of the onlysignificant open reading frame in the clone (at residues 51-66); thepredicted amino acid sequence reveals that position 10 of the maturepeptide (residue 60 of the precursor) is encoded by a codon forthreonine. Thus, the Edman sequencing, together with cloning results,suggested that a modified threonine residue was present in position 10.TABLE 1 DNA (SEQ ID NO:5) and Peptide (SEQ ID NO:6) Sequences ofContulakin-G atg cag acg gcc tac tgg gtg atg gtg atg atg atg Met Gln ThrAla Tyr Trp Val Met Val Met Met Met gtg tgg att gca gcc cct ctg tct gaaggt ggt aaa Val Trp Ile Ala Ala Pro Leu Ser Glu Gly Gly Lys ctg aac gatgta att cgg ggt ttg gtg cca gac gac Leu Asn Asp Val Ile Arg Gly Leu ValPro Asp Asp ata acc cca cag ctc atg ttg gga agt ctg att tcc Ile Thr ProGln Leu Met Leu Gly Ser Leu Ile Ser cgt cgt caa tcg gaa gag ggt ggt tcaaat gca acc Arg Arg Gln Ser Glu Glu Gly Gly Ser Asn Ala Thr aag aaa ccctat att cta agg gcc agc gac cag gtt Lys Lys Pro Tyr Ile Leu Arg Ala SerAsp Gln Val gca tct ggg cca tag Ala Ser Gly Pro

[0109] Mass spectrometric analyses (MALD, LSI and nano-ESI) of thepurified contulakin-G fraction revealed a variety of intact species assummarized in Table 2. Some variation in the intensity of the differentspecies was observed with different ionization techniques, which wasascribed to differences in the bias (Craig et al., 1994) with eachionization technique. In the following analysis, we have concentrated onthe major glycoform with intact mass M₁=2069 observed with all of theionization techniques investigated. The difference between the observedmass (2069 Da) and the mass calculated for the sequence assuming Thr atresidue 10 (1703.83 Da) was 365 Da. Because one possible modification ofthreonine is O-glycosylation, we proposed, based on this massdifference, that the unidentified residue washexose-N-acetyl-hexosaminethreonine (Hex-HexNAc-Thr) which would resultin the addition of 365.13 Da. The observed masses (Table 2) areconsistent with the calculated monoisotopic mass of the [M₁+H]⁺ or[M₁+2H]²⁺ of the proposed disaccharide-linked peptide (2069.98 or 1035.5Da respectively). Intense fragment ions were observed in the nano-ESIMS/MS mass spectrum of the doubly charged [M₁+2H]²⁺ intact molecule ionof contulakin-G (FIG. 6) corresponding to the loss of the completeHex-HexNAc glycan (denoted p(χ₃)₁₀ (Craig et al., 1993) or loss of theterminal hexose residue (P(χ₈)₁₀). TABLE 2 Species Observed with MALD,LSI and nano-ESI Analysis of Purified Contulakin-G Molecule M₁ (Da) M₂(Da) M₃ (Da) M₄ (Da) Species Proposed HexHexNAc SO₄HexHexNAc Hex₃Hex₂HexNAc₂ glycan MALD/TOF 2069^(a) — 2186^(b) — LSI/ 2068.7 2149.6 —2436.5 Magnetic nano-ESI/IT 2068.6^(c) 2148.6^(c) — — Mono^(d) 2068.972148.92 2189.94 2434.10 [M + H]⁺ Average^(e) 2070.19 2150.25 2191.272435.53 [M + H]⁺

Example 4

[0110] Evidence that Thr-10 is O-glycosylated

[0111] Native contulakin-G was treated with β-galactosidase isolatedfrom bovine testes. This enzyme preferentially hydrolyzes terminal β1->3galactopyranosyl residues from the non-reducing end of glycoconjugates.After β-galactosidase treatment of the native sample a new component wasobserved on RP-HPLC. This component was collected and analyzed withMALD-MS in which a species was observed at m/z 1907. The difference inmass and the specificity of the enzyme are consistent with a terminalgalactose residue being released. Based on the β-galactosidasehydrolysis results we reasoned that the glycan moiety might besusceptible to O-glycosidase treatment, which liberates the disaccharideGal (β1->3) GalNAc (α1->) bound to serine or threonine as a core unit ofglycopeptides. O-glycosidase treatment of the native contulakin-G did infact result in a new species after the enzyme hydrolysis mixture wasanalyzed on RP-HPLC. The new component was collected and analyzed withMALD-MS where an m/z 1704 species was observed consistent with loss ofHex-HexNAc (i.e., the mass was consistent with that predicted for thepeptide with an unmodified threonine residue at position 10). The enzymehydrolysis results are consistent with the presence of a Gal (β1->3)GalNAc (α1->) glycan. Based on the O-glycosidase and the P-galactosidasehydrolysis results, the structure of the most abundant glycopeptide is:                Gal (β1->3) GalNAc (α1->) (SEQ ID NO:1)                                     |pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH

Example 5 Synthesis of the Non-Glycosylated and GlycosylatedContulakin-G

[0112] The 16-amino acid non-glycosylated peptide was chemicallysynthesized. The synthetic material was found to have the same retentiontime as the enzymatically des-glycosylated contulakin-G on RP-HPLC. The16-amino acid glycosylated contulakin-G containing Gal(β1->3) GalNAc(α1->) attached to Thr₁₀ was also synthesized. This syntheticglycosylated contulakin-G co-eluted with the native contulakin-G onRP-HPLC. The post source decay fragmentation spectra observed for bothnative and synthetic contulakin-G showed very similar fragmentationpatterns.

Example 6 Biological Potency of Synthetic Glycosylated andNon-Glycosylated Contulakin-G

[0113] The loss of motor control for which the native contulakin-G wasoriginally isolated, together with gut contraction, absence ofpreening/grooming, and reduced sensitivity to tail depression were signsobserved when Neurotensin₁₋₁₃, non-glycosylated Thr₁₀-contulakin-G orsynthetic contulakin-G were administered icv. In order to investigatethese observations in more detail, a dose response comparison wasperformed as detailed in Table 3. While the non-glycosylatedThr₁₀-contulakin-G analog was active at doses of 1 nmol and higher, itwas inactive at 300 pmol doses. In contrast, contulakin-G was found toelicit loss of motor control at doses of 30 pmol or approximately 5pmol/g. TABLE 3 Effect of icv administration of Neurotensin₁₋₁₃,Thr₁₀-Contulakin-G and Contulakin-G in 14-18 day old mice Av. Time ofsymptom dose Number age weight A^(c) B^(c) R^(c) Compound (pmol) ofmice^(a) (days) (g)^(b) (min) (min) (min) NSS 0 8 16 6.2 —^(d) —^(d)—^(d) neuro- 1000 2 14 7.1 9 23 >120 tensin₁₋₁₃ Thr₁₀-contu- 1000 7 16.66.5 9 99 159 lakin-G Thr₁₀-contu- 300 6 15.7 6.1 —^(d) —^(d) —^(d)lakin-G contulakin-G 1000 6 18 7.1 1.0 120 187 contulakin-G 300 8 15.56.6 2.9 42 151 contulakin-G 100 8 15.9 6.6 2.8 40 136 contulakin-G 307/9 15 6.3 5.3 23 114 # when a mouse was placed onto a bench top afterlifting it by the tail for a second. Symptom A: The mouse moved at mosta few steps and rested with the hind legs spread out. # Mouse remainedstationary unless pushed or lifted. Symptom B: The mouse remainedsluggish but the position of the hind part of the body when at restresembled that of the NSS # controls. Symptom R: Recovery, the mousemoved freely when released.

[0114] The six C-terminal amino acids of contulakin-G show significantsimilarity to the sequences of neurotensin₁₋₁₃, neuromedin, xenin andthe C-terminus of xenopsin (see Table 4). Because of the similar signsobserved when either contulakin-G or Neurotensin₁₋₁₃ were administeredicv and the significant homology between contulakin-G andNeurotensin₁₋₁₃, we tested the affinity of contulakin-G for a number ofthe cloned neurotensin receptors. As shown in Table 5, thenon-glycosylated Thr₁₀-contulakin-G analog was found to bind the humanneurotensin type I receptor (hNTR1) with 10 fold lower affinity thanNeurotensin₁₋₁₃, and even lower affinities for the other NTR's.Contulakin-G exhibited significantly lower affinity than thenon-glycosylated Thr₁₀-ocontulakin-G analog for all of the NTR's tested.

[0115] Both contulakin-G and the non-glycosylated Thr₁₀-contulakin-Ganalog acted as agonists when tested on CHO cells expressing the rNTR1.No response was observed with CHO cells expressing the rNTR2. Thenon-glycosylated Thr₁₀-contulakin-G analog resulted in slightly lowerpotency (0.6 nM) but with similar efficacy as compared withNeurotensin₁₋₁₃. The synthetic glycosylated contulakin-G potency wassignificantly lower (20-30 nM) and the agonistic efficacy wasapproximately half that observed for Neurotensin₁₋₁₃. TABLE 4 SequenceComparison of Contulakin-G and Members of the Neurotensin Family ofPeptides Name Sequence (SEQ ID NO:) Id^(a) Si^(b) Source Ref Contulakin-<ESEEGGSNAT*KKPYIL-OH — — C. geographus G (7) neurotensin<ELYENKPRRPYIL-OH 66 33 bovine (1) (8) hypothalamus neuromedin KIPYIL-OH83  0 porcine spinal cord (2) N (9) xenopsin QGKRPWIL-OH 66 16 Xenopuslaevis (3) (10) xenin MLTKFETKSARVKGLSFHPKRPWIL-OH 66 16 human gastric(4) (12) mucosa

[0116] References (1) Carraway et al., 1973; (2) Minamino et al., 1984;(3) Araki et al., 1973; (4) Feurle et al., 1992. TABLE 5 Comparison ofBinding Affinity of Neurotensin_(1—13), Thr₁₀-Contulakin-G andContulakin-G for the Cloned Human and Rat Neurotensin Type 1 Receptor(NTR1), the Rat Neurotensin Type 2 Receptor (rNTR2), and the SolubilizedMouse Neurotensin Type 3 Receptor (mNTR3) IC₅₀ (nM) Receptor CompoundhNTR1 rNTR1 rNTR2 mNTR3 neurotensin_(1—13) 1.4 3.2 6.0 1.4Thr₁₀-contulakin-G 23 79 170 71 contulakin-G 960 524 730 250

Example 7 Biological Activity of Contulakin-G Analogs

[0117] The biological activity of several peptide analogs ofcontulakin-G was tested in a similar manner as described above by icvinjection in mice. These peptides were synthesized as described herein,and include the following analogs:

[0118] Ser₁₀-contulakin-G containing the native glycosylation on Ser₁₀(analog A); and

[0119] Δ1-9-Ser₁₀-contulakin-G containing the native glycosylation onSer₁₀ (analog B)

[0120] It was found that analog A was slightly more active than thenative contulakin-G. It was also found that analog B had the sameactivity, i.e., onset and recovery time than analog A when tested in twoweek old mice at a dose of 100 pmole. In this test, the mice were stillnot able to right themselves after 75 minutes. When tested in three weekold mice at doses of 1 nmole and 300 pmole, the same activity was seenbetween the analogs and these mice were drowsy for 100 minutes. Theseexperiments demonstrate that glycosylated contulakin-G analogs in whichN-terminal amino acids residues have been removed, retain activity.Similar results are achieved for other analogs, such asΔ1-5-Ser₆-contulakin-G containing the native glycosylation on Ser₆ withor without the native glycosylation on Thr₁₀. These results show thatthe placement of a glycosylated serine residue proximal to the site oftruncation yields active analogs.

[0121] The Conus peptide characterized above, contulakin-G, has a novelbiochemical feature: a post-translationally O-glycosylated threonine notpreviously found in Conus peptides. Using mass spectrometry and specificenzymatic hydrolyses, it was found that Thr₁₀ was modified with thedisaccharide Gal (β1->3) GalNAc (α1->). The corresponding glycosylatedand non-glycosylated forms of contulakin-G were synthesized whichconfirmed the molecular structure of this major glycosylated form of thenative molecule based on RP-HPLC co-elution and MS fragmentationcriteria. The masses of the other more minor molecule species observedwith mass spectrometry are consistent with glycan structural variationsat peripheral sites on the characterized oligosaccharide core unit(Baenziger, 1994).

[0122] An analysis of a cDNA clone encoding contulakin-G reveals thatthe prepropeptide organization of the contulakin-G precursor is similarto that of other Conus peptide precursors (Olivera et al., 1997). Atypical signal sequence is found, and immediately N-terminal to thecontulakin-G sequence are two basic amino acids which presumably signala proteolytic cleavage to generate the N-terminus of the mature peptide(the glutamine residue would cyclize to pyroglutamate eitherspontaneously or due to the action of glutaminyl cyclase (Fischer etal., 1987)). Although in most respects the contulakin-G precursor hasthe same organization as all other Conus venom peptide precursors andwould be predicted to be processed in the same way, the ten C-terminalamino acids predicted by the clone are not present in contulakin-Gpurified from venom. One possibility is that the clone represents adifferent variant, for example one which was alternatively spliced.Alternatively, furtherproteolytic processing at the C-terminus mayberequired to generate mature contulakin-G.

[0123] Over the last 20 years an increasing number of biologicallyimportant glycopeptides and glycoproteins have been identified.Vespulakinin 1, first identified by Pisano et al. (Yoshida et al.,1976), is, to our knowledge, the only other O-glycosylated peptide toxinwhich has been isolated from venom other than Conus. Vespulakinin 1 wasextracted from the venom sacs of the yellow jacket wasp, Vespulamaculifrons. The peptide (TAT*T*RRRGRPPGFSPFR-OH (SEQ ID NO: 12) wherethe asterisk indicates an O-linked glycosylated threonine residue)contains two sequential sites of O-linked glycosylation. The C-terminusof Vespulakinin is identical to the sequence of Bradykinin (RPPGFSPFR-OH(SEQ ID NO: 13)) and the peptide was found to elicit a number of signsalso elicited by Bradykinin. Vespulakinin is therefore another exampleof an O-linked glycosylated peptide toxin in which the C-terminusappears to target a mammalian neurotransmitter receptor. Thus, bothcontulakin-G and Vespulakinin I contain glycosylated N-terminalextensions to sequences with very high homology to mammalianneuropeptides. κA-conotoxin SIVA, a K⁺ channel inhibitor is unusualamong disulfide-rich Conus peptides in having a long N-terminal tail,which has an O-glycosylated residue (Craig et al., 1998).

[0124] For most Conus peptides, a specific conformation appears to bestabilized either by multiple disulfide linkages or by the appropriatespacing of γ-carboxyglutamateresidues to promote formation of α-helices(Olivera et al., 1990). Conus peptides without multiple disulfidescomprise a most eclectic set of families, including the conopressins,conantokins, contryphans and now contulakin-G. The conopressins areprobably endogenous molluscan peptides, clearly homologous to thevassopressin/oxytocin family of peptides; these are more widelydistributed in molluscan tissues than in Conus venom ducts. However, theother non-disulfide-rich peptides (conantokins, contryphans andcontulakin-G) may be specialized venom peptides exhibiting unusualpost-translational modifications. In addition to the O-glycosylatedthreonine moiety of contulakin-G described here, γ-carboxylation ofglutamate residues and the post-translational epimerization andbromination of tryptophan residues were discovered in conantokins andcontryphans.

[0125] Several lines of evidence are consistent with contulakin-G beingthe first member of the neurotensin family of peptides to be isolatedfrom an invertebrate source. First, the C-terminal region ofcontulakin-G exhibits a striking degree of similarity to other membersof the neurotensin family (all from vertebrates), as shown in Table 4.Furthermore, it was shown above that contulakin-G competes for bindingto three known neurotensin receptor subtypes; evidence that contulakin-Gacts as an agonist on a cloned neurotensin receptor is also presentedabove. Most convincingly however, when contulakin-G is injected intomice, the same behavioral signs are elicited with administration ofneurotensin. Thus, structural data, binding data and in vivo behavioralsymptomatology are all consistent with the assignment of contulakin-G tothe neurotensin family of peptides.

[0126] Clearly, both contulakin-G and the non-glycosylatedThr₁₀-contulakin-G are rNTR1 agonists at physiologically relevantconcentrations (20-30 and 0.6 nM, respectively). The observed agonisticeffects of both contulakin-G and the non-glycosylated analog, as well asthe absence of any agonistic effect of these ligands on CHO cellsexpressing rNTR2 using the IP accumulation assay does not correlate withthe in vitro binding data; both peptides are agonists at concentrationssignificantly below their IC₅₀ binding affinity (524 and 79 nM,respectively). Most unexpected therefore, given its apparently lowerbinding affinity, is the increased potency of glycosylated contulakin-Gcompared with the non-glycosylated analog after icv administration.

[0127] Thus, the role of the glycan is somewhat paradoxical. In vitro,the glycan neither increases the binding affinity, the agonistic potencynor agonistic efficacy. In contrast, in vivo, the glycan significantlyincreases the potency of the peptide. One simple explanation is that theincreased potency of contulakin-G compared with the non-glycosylatedanalog is due to increased stability. An alternative mechanism for theincreased potency is transport to the site of action facilitated by theglycan. Additionally, the glycosylated peptide may act with highaffinity on an as-yet-undefined neurotensin receptor subtype (Tyler etal., 1998), or may be a selective high affinity ligand for a particularstate of a neurotensin receptor subtype. Yet another possibility is thatthe relevant targeted neurotensin receptors may be closely co-localizedwith carbohydrate binding sites, and that the glycan may serve as an“address label”, a mechanism postulated for certain opiate peptides.Preliminary data supporting the increased stability hypothesis has beenobtained—proteolytic degradation of contulakin-G is inhibited by thepresence of the glycan moiety. The increased stability may well resultin an enhanced supply of the glycopeptide at the receptor. However, theincreased in vivo potency of contulakin-G conferred by O-glycosylationclearly requires a more balanced evaluation of the possibilitiesoutlined above.

Example 8 Materials and Methods for Assessing

[0128] 1. Analgesic Activity of Thr₁₀-Contulakin-G

[0129] 1. Acute pain (hotplate). Thr₁₀-contulakin-G (CGX-1063) orvehicle was administered via intracerebroventricular (icv) in a volumeof 5 μl. Fifteen minutes after injection, animals were placed on a 55°C. hotplate. The latency to the first response (flinch), a spinallymediated behavioral response, and the first hindlimb lick, a centrallyorganized motor response to acute pain, were recorded. Mice were removedfrom the hotplate after 60 seconds if no response was observed.Immediately prior to being placed on the hotplate, motor function wastested by determining the latency to first fall from an acceleratingrotarod.

[0130] 2. Persistentpain (formalin test). Intrathecal (it) druginjections were performed as described by Hyldon and Wilcox (1980).CGX-1063 (10 or 100 pmol) or vehicle was administered in a volume of 5μl. Fifteen minutes after the it injection, the right hindpaw wasinjected with 20 μl of 5% formalin. Animals were placed in clearplexiglass cylinders backed by mirrors to facilitate observation.Animals were closely observed for 2 minutes per 5 minute period, and theamount of time the animal spent licking the injected paw was recorded inthis manner for a total of 45-50 minutes. Results are expressed aslicking time in seconds per five minutes. At the end of the experiment,all animals were placed on an accelerating rotorod and the latency tofirst fall was recorded.

[0131] 2. Neuropathic pain. The partial sciatic nerve ligation model wasused to assess the efficacy of CGX-1063 in neuropathic pain. Nerveinjury was produced according to the methods of Malmberg and Basbaum(1998). Animals were anesthetized with a ketamine/xylazine solution, thesciatic nerve was exposed and tightly ligated with 8-0 silk suturearound ⅓ to ½ of the nerve. In sham-operated mice the nerve was exposed,but not ligated. Animals were allowed to recover for at least 1 weekbefore testing was performed. On the testing day, mice were placed inplexiglass cylinders on a wire mesh frame and allowed to habituate forat least 60 minutes. Mechanical allodynia was assessed with calibratedvon Frey filaments using the up-down method as described by Chaplan etal. (1994), and the 50% withdrawal threshold was calculated. Animalsthat did not respond to any of the filaments in the series were assigneda maximal value of 3.6 grams, which is the filament that typicallylifted the hindlimb without bending, and corresponds to approximately{fraction (1/10)} the animal's body weight.

Example 9 Analgesic Activity of Thr₁₀-Contulakin-G

[0132] CGX-1063(10 fmol-10 nmol, icv) dose-dependently increased thelatency to the first hindpaw lick and first response elicited by thehotplate (FIGS. 7A-7B). Of interest is the difference in potency ofCGX-1063 in increasing the latency to the first hindpaw lick compared tothe latency to first response. CGX-1063 also dose-dependently decreasedthe latency to first fall on the rotarod (FIG. 7C). However, thisapparent motor impairment did not appear to be the result of the loss ofmotor function, since animals were capable of normal locomotor activitywhen stimulated. Thus, the effect of CGX-1063 on the hotplateunequivocally was an analgesic effect.

[0133] CGX-1063(10 or 100 pmol, it) dose-dependently and significantlydecreased the second phase of the formalin test (FIG. 8A).Interestingly, the lower dose (10 pmol) was more effective in decreasingthe first phase response time than was the higher dose. This will beexamined in more detail in future experiments. After it administration,CGX-1063 treated animals showed no motor impairment compared to vehicletreated animals (FIG. 8B), indicating that the effect of icv CGX-1063(observed in the hotplate test above) on motor impairment is mediated athigher brain regions, not spinally, and that the analgesic effects ofCGX-1063 can be separated from the motor toxicity by using this route(it) of administration. The downward shift in the rotorod scorescompared to those from animals used in the hotplate test reflects anoverall impairment in these animals due to formalin-induced allodyniaand inflammation of the hindpaw.

[0134] One week after partial sciatic nerve ligation, animals showed amarked decrease in the paw withdrawal threshold on the operated side(ipsilateral) relative to the unoperated side (contralateral),indicating an increase in sensitivity to mechanical stimuli (FIG. 9).Intrathecal administration of CGX-1063 (100 pmol) dramatically increasedthe withdrawal threshold on the ligated side (an approximate six foldincrease). Interestingly, the mechanical threshold on the contralateralside was not significantly altered. In sham-operated animals, there wasno difference in withdrawal threshold between operated and un-operatedsides. After intrathecal CGX-1063, the withdrawal threshold wasuniformly increased in both hindpaws of these animals.

[0135] The present data demonstrate that CGX-1063 has potent analgesicproperties in three commonly used models of pain: acute,persistent/inflammatory and neuropathic pain models. CGX-1063administered centrally (icv) dose-dependently reduced the responselatency in the hot plate model of acute pain, and was effective in thelow picomole to high femtomole range. Preliminary data indicate that theanalgesic effect of CGX-1063 in this model is not mediated through anopioid mechanism. CGX-1063 was also effective in reducing nociceptiveactivity in the formalin model of persistent/inflammatory pain. CGX-1063dose-dependently reduced the second (inflammatory) phase of the formalintest, while at the lower dose, reduced phase one activity. Finally,CGX-1063 showed profound analgesic activity in a model of neuropathicpain. Mechanical withdrawal thresholds in this model were increasednearly six fold compared to pre-treatment values, while not alteringsensitivity in the non-injured paw, possibly indicating that CGX-1063reduces neuropathic allodynia while not affecting normal sensorytransmission.

Example 10 P Materials and Methods for Assessing Analgesic Activity ofContulakin-G

[0136] 1. Acute pain (tail-flick). Drug (contulakin-G (CGX-1160) orThr₁₀-contulakin-G (CGX-1063)) or saline was administered intrathecally(i.t.) according to the method of Hylden and Wilcox (Hylden and Wilcox,1980) in a constant volume of 5 μl. Mice were gently wrapped in a towelwith the tail exposed. At various time-points following the i.t.injection, the tail was dipped in a water bath maintained at 54° C. andthe time to a vigorous tail withdrawal was recorded. If there was nowithdrawal by 8 seconds, the tail was removed to avoid tissue damage.

[0137] 2. Persistent pain formalin test). CGX-1160, CGX-1063 (1, 10 or100 pmol), neurotensin (NT) (1, 10, 100 or 10000 pmol), or vehicle wasadministered i.t. in a volume of 5 μl. Fifteen minutes after the i.t.injection, the right hindpaw was injected with 20 μl of 5% formalin.Animals were placed in clear plexiglass cylinders backed by mirrors tofacilitate observation. Animals were closely observed for two minutesper five minute period, and the amount of time the animal spent lickingthe injected paw was recorded in this manner for a total of 45-50minutes. Results are expressed as licking time in seconds per fiveminutes. At the end of the experiment, all animals were placed on anaccelerating rotorod and the latency to first fall was recorded.

[0138] 3. Chronic inflammatory allodynia (CFA model). Mice were givenintraplantar (i.pl.) injections of 20 μl of CFA into the right hindpawand returned to their home cage. Three days later mice were placed inplexiglass cylinders on a wire mesh frame and allowed to habituate forat least 60 minutes. Mechanical allodynia was assessed with calibratedvon Frey filaments using the up-down method as described (Chaplan etal., 1994), and the 50% withdrawal threshold was calculated. Animalsthat did not respond to any of the filaments in the series were assigneda maximal value of 3.6 grams, which is the filament that typicallylifted the hindlimb without bending, and corresponds to approximately{fraction (1/10)} of the body weight.

[0139] 4. Toxicity testing. To accurately assess the motor impairingeffects of CGX-1160, CGX-1063, and NT, 50 mice were divided into groupsreceiving i.t. CGX-1160 or CGX-1063 (1, 10, 100, 500 and 1000 pmol), NT(0.1, 1, 10, and 100 nmol), or saline (n=5 per group except for thehighest dose of each compound where n=3). Starting at 15 minutes postinjection animals were place on an accelerating rotorod and the latencyto first fall was recorded. Animals were retested at 30, 60, 120, 240and 300 minutes (or until the latency to fall had returned to controlvalues). Rectal temperature was also recorded in these animals at thesame time points.

Example 11 Analgesic Activity of Contulakin-G

[0140] CGX-1160 dose-dependently increased the tail-flick latency (FIG.10A) with a time to peak effect of ≦30 minutes (the earliest timetested, FIG. 10B). Furthermore, the increase in latency was long-lastingwith elevated withdrawal times at 5 hour post injection that returned tobaseline at 24 hours post injection (FIG. 10B). CGX-1063 also showed adose-dependent, though more variable increase in withdrawal latency, andshowed only modest antinociceptive efficacy in this model relative toCGX-1160 (FIGS. 10A-10B). In comparison, NT did not significantlyelevate withdrawal latency in the tail-flick assay (FIGS. 10A-10B).

[0141] All of the compounds tested dose-dependently showedantinociceptive properties in both phases of the formalin test, but withdifferent potencies. CGX-1160 was the most potent of the threecompounds. In phase 1 of the formalin test (FIG. 1A), CGX-1160 had anED₅₀ of approximately 30-40 pmol while NT had an ED₅₀ of ≈1 nmol.CGX-1063 did not reach the 50% antinociception threshold in phase 1,however, the irregular dose-response in this test warrants repeating the100 pmol dose in this assay. In phase 2 of the formalin test, all threecompounds dose-dependently reduced the paw licking time (indicated inthe figures as an increase in the percent antinociception; FIG. 1B).Again, CGX-1160 was more potent than the other compounds with anestimated ED₅₀ of 1 pmol. Lower doses of this compound will be assessedin the future to complete the dose response curve necessary to calculatea more precise ED₅₀. CGX-1063 was also effective in reducing nociceptivebehavior in phase 2, with an estimated ED₅₀ of 10-20 pmol. NT wasdramatically less potent than either of the contulakins with anestimated ED₅₀ of 600-700 pmol (FIG. 11B).

[0142] CGX-1160 showed extremely potent and dose-dependent reversal ofCFA-induced mechanical allodynia (FIG. 12A). One-hundred (100) fmol ofCGX-1160 given i.t. completely reversed the CFA-induced mechanicalallodynia. Interestingly, at this dose, the contralateral sensitivity tomechanical pressure was unaltered indicating a potential unilateralalteration in NT receptors in chronic inflammation. At higher doses ofCGX-1160, the mechanical withdrawal threshold in both the CFA-injectedpaw and the contralateral uninjected paw was dramatically elevated. InFIGS. 12A and 12B, the numbers over the bars indicate the percentincrease in mechanical threshold relative to the pre-drug level. Asindicated, at all doses tested, CGX-1160 had a much greaterantiallodynic effect on the CFA injected side relative to the uninjectedside. CGX-1063 was less potent than CGX-1160, but also completelyreversed the CFA-induced allodynia (FIG. 12B). The minimally effectivedose was 10 pmol, however, at this dose, unlike CGX-1160, thecontralateral side was also elevated relative to pre-drug baselinemeasurements. Consistent with the other models examined in this study,NT showed efficacy in the CFA model at 1 mol, but not at 100 pmol (FIG.12C). Other doses of CGX-1160 and NT will be examined in the future todetermine accurate ED₅₀s for these compounds.

[0143] CGX-1160, -1063, and NT all showed dose-dependent effects onlocomotor impairment and body temperature. For all three compounds,maximal impairment was at 15 minutes post i.t. injection (locomotorimpairment, FIG. 13A) or 30 minutes (hypothermic effects, FIG. 14A).CGX-1063 had no motor toxicity at the lowest dose tested (1 pmol, FIG.13B), but at higher doses animals showed significant motor toxicity(estimated TD₅₀ of 10 pmol, FIGS. 13B and 15A). At 10 pmol this toxicitylasted for 30 minutes, but resolved by 60 minutes. When 100 pmol or 1nmol was administered, animals were motor impaired for 2-3 hours (FIG.14A). CGX-1160 was equipotent to CGX-1063 in causing motor impairment(estimated TD₅₀ of 10-20 pmol, FIG. 13B). Similar to CGX-1063, at higherdoses (100-500× its ED₅₀) CGX-1160 showed motor impairment that resolvedafter 5 hours (FIGS. 13A and 14B). The estimated TD₅₀ for NT-inducedmotor impairment was 3 nmol (FIG. 13B). Similar to the contulakins, athigh doses, NT-induced motor impairment that lasted 2-4 hours (FIGS. 13Aand 14C).

[0144] The hypothermic effects of these compounds were similar to motortoxicity. All three caused a dose-dependent decrease in bodytemperature. CGX-1160 and -1063 were equipotent with an estimated TD₅₀of 100 pmol (FIG. 15B). However, at this dose CGX-1063 induced a drop inbody temperature lasting 2-3 hours (FIGS. 15A and 16A), while thehypothermic effect caused by CGX-1160 resolved by 60 minutes (FIGS. 15Aand 16B). At the highest dose of CGX-1160 (500 pmol, 500× the ED₅₀), thehypothermic effect had not resolved by six hours post-injection (FIG.16B). NT showed a very similar dose-response and time course to thecontulakins. At the lower doses, NT had no effect or showed a shortlasting hypothermic effect (FIG. 16C). At the highest dose, however (100nmol), NT caused a dramatic and long-lasting hypothermia that had notresolved by three hours (FIGS. 15A and 16C).

[0145] The present data show that CGX-1160 and CGX-1063 are potent,broad-spectrum analgesic agents effective in several animal models ofacute and chronic pain. CGX-1160 is typically 10 fold more potent thanCGX-1063, and 1000 times more potent than NT (Table 6). CGX-1160 isparticularly potent in the model of chronic inflammatory pain whereCGX-1160 selectively increases the mechanical withdrawal threshold onlyin the paw receiving the CFA injection, while not altering the thresholdof the uninjected paw. This finding indicates that chronic inflammationmay lead to a reorganization of NT receptors in nociceptive pathwayscorresponding to the inflamed paw. Since CGX-1160 was the only compoundin these experiments to show an increased potency, this may indicate anupregulation of a receptor subtype for which CGX-1160 may haveparticular selectivity and specificity. In support of this hypothesis ofCGX-1160 subtype selectivity are the findings that this compound showsantinociception at doses 10-100 fold less than for either locomotorimpairment or hypothermia, whereas CGX-1063 and NT causeantinociception, locomotor impairment, and hypothermia at approximatelyequal doses when administered i.t. Particularly interesting is thelong-lasting hypothermic effect of CGX-1063. When given i.t. at 100 pmol(approximately 10 times its ED₅₀ in phase 2 of the formalin test, seeFIG. 16A), CGX-1063 caused long-lasting hypothermia relative tocomparable antinociceptive doses of CGX-1160 (compare the 10 pmol dosein FIG. 16B) and NT (compare the 10 nmol dose in FIG. 16C). Thispotentially indicates that CGX-1063 is selective for the NT receptorsubtype involved in the hypothermic effect of NT analogs. Thus theO-glycosylation of Thr₁₀ in CGX-1160 may impart selectivity for theantinociceptive NTR subtype, currently thought to be NTR2, as well asmetabolic resistance to peptidases. TABLE 6 Comparison of theAntinociceptive Effects, Motor Impairment Effects, and Protective Indexof CGX-1160, CGX-1063, and NT in the Formalin Test (phase 2) andCFA-Induced Allodynia Test Compound ED₅₀, pmol TD₅₀, pmol PI FormalinTest (phase 2) CGX-1160 1 10-20 10-20 CGX-1063   10-20 10-20 1-2 NT  600-700 3000   5-4.3 CFA-Induced Allodynia Test CGX-1160 <0.110-20 >100 CGX-1063 <10 10-20 1-5 NT ≈500-600 3000 5-6

Example 12 Materials and Methods for Assessing Antipsychotic Activity ofContulakin-G

[0146] 1. Materials. D-amphetamine was obtained from Sigma (St. Louis,Mo.). Contulakin-G (CGX-1160; a synthetic 16 amino acid O-linkedglycopeptide) was synthesized as described above.

[0147] 2. Animals. Male CF-1 mice (30-35 g; Charles River Laboratories)were used. All animals were housed in a temperature controlled (23°±3°C.) room with a 12 hour light-dark cycle with free access to food andwater. All animals were euthanized in accordance with Public HealthService policies on the humane care of laboratory animals.

[0148] 3. Locomotor Activity. Animals were placed in clear plastic cages(40 cm×22 cm, 20 cm deep) and allowed to acclimate for 30 minutes.Animals then received either contulakin-G (100 pmol) or saline (vehicle)by freehand intracerebroventricular (i.c.v.) injection (5 μl volume)through a 10 μl Hamilton syringe. After 5 minutes, animals receivedsaline or D-amphetamine sulphate (3 mg/kg) via intraperitoneal (i.p.)administration. Distance traveled (cm) and time spent ambulatory (s)were monitored for 30 minutes using a Videomex-V tracking system(Columbus Instruments, Columbus, Ohio). All testing was done in anisolated, dimly lit behavioral room.

[0149] 4. Statistics. Data were analyzed using one-way analysis ofvariance (ANOVA) with drug treatment as the only factor, followed by aNewman-Keuls multiple comparison test for comparison of individualgroups, with P<0.05 accepted as statistically significant. Statisticalanalyses were performed with GraphPad PRISM software (Version 2.01,GraphPad, San Diego, Calif.).

Example 13 Antipsychotic Activity of Contulakin-G

[0150] A significant effect of drug treatment on locomotor activity asmeasured by both distance traveled [F(4,21)=7.87, P<0.05] and time spentambulating [F(4,21)=6.17, P<0.05] was found in the present study.Administration of D-amphetamine resulted in a dose dependent increase inboth distance traveled and time spent ambulating (FIGS. 17-18).Pretreatment of mice with contulakin-G (100 pmol i.c.v.) significantlyreduced amphetamine-stimulated (3 mg/kg i.p.) increases in distancetraveled and time spent ambulating. A reduction in basal locomotoractivity (both distance traveled and time spent ambulating) was seenafter pretreatment with contulakin-G (100 pmol i.c.v.), however, thisreduction did not reach statistical significance.

[0151] Converging lines of evidence imply that neurotensin may haveantipsychotic properties without the associated adverse side effectprofiles of standard neuroleptic drugs (reviewed in (Nemeroff et al.,1992)). Subsequently, many groups have focused on neurotensin analogs asnovel antipsychotic drugs. Since contulakin-G shares C-terminal homologywith neurotensin, and resembles neurotensin in both in vivo and in vitroassays, the ability of contulakin-G to inhibit D-amphetamine-stimulatedlocomotor activity, a preclinical screen predictive of antipsychoticefficacy, was assessed. This example demonstrates that pretreatment ofmice with contulakin-G significantly reduced amphetamine-stimulatedincreases in locomotor activity. These data indicate that contulakin-Ghas similar antipsychotic activity as neurotensin. However as shownabove, while neurotensin was far more potent than contulakin-G at therat neurotensin receptors rNTR1 (IC₅₀: 3.2 nM for neurotensin; 524 nMfor contulakin-G) and rNTR2 (IC₅₀: 6.0 nM for neurotensin; 730 nM forcontulakin-G), and the mouse neurotensin receptor mNTR3 (C₅₀: 1.4 nM forneurotensin; 250 nM for contulakin-G), contulakin-G was 1 to 2 orders ofmagnitude more potent in an in vivo assay (a visually rated assessmentof locomotor activity) following i.c.v. administration. These resultsindicate that contulakin-G and neurotensin may interact with overlappingbut distinct populations of neurotensin receptor subtypes or activationstates. Thus, contulakin-G would not share the limiting side effects ofneurotensin.

Example 14 Materials and Methods for Assessing Anticonvulsant Activityof Contulakin-G

[0152] 1. Animals. Male Frings (20-25 g) were housed in a temperaturecontrolled (23°±1° C.) room with a 12 hour light-dark cycle with freeaccess to food and water. Mice were housed, fed, and handled in a mannerconsistent with the recommendations in HEW publication (NIH) No. 8623,“Guide for the Care and Use of Laboratory Animals.” All mice wereeuthanized in accordance with Public Health Service policies on thehumane care of laboratory animals.

[0153] 2. Anticonvulsant Assessment. Frings mice were placed in a round,plexiglass jar (diameter 15 cm, height 18 cm) and exposed to a soundstimulus of 110 decibels (11 KHz). Mice were then observed for 25 secfor the presence or absence of hindlimb tonic extension. Animals notdisplaying hindlimb tonic extension were considered protected.

[0154] 3. Rotorod Test. Motor impairment was assessed at time of peakeffect by placing mice on a rotorod turning at 6 rpm. Animals fallingthree times in one minute were considered impaired.

Example 15 Anticonvulsant Activity of Contulakin-G

[0155] Contulakin-G (CGX-1160) and Thr₁₀-contulakin-G (CGX-1063)potently and dose-dependently blocked audiogenic seizures in Frings micefollowing i.c.v. administration (FIG. 19). Similar to the efficacy inpain models, CGX-1160 was more potent than CGX-1063 with ED₅₀s of 7.1pmol and 27.0 pmol, respectively (Table 7). Also consistent withprevious studies, NT was dramatically less potent than CGX-1160 or-1063. Although a dose-response curve for NT has not yet been completed,NT showed 50% protection following 1 nmol administered i.c.v. Whentested for motor toxicity, CGX-1160 did not reach the 50% toxic level atdoses up to 200 pmol (FIG. 19), whereas the TD₅₀ for CGX-1063 isestimated to be approximately 375 pmol resulting in an estimated PI of14 for the doses tested. TABLE 7 Anticonvulsant Profile of CGX-1160 andCGX-1063 in Frings AGS Mice Following i.c.v. Administration Time of testCompound (min.)^(a) TD₅₀(pmol) ED₅₀(pmol) P.I.^(b) X more potent than NTCGX-1160 15, 60 >200  7.1 >28 ≈140 (4.9-8.5) CGX-1063 15, 60 ≈375 27.0≈14 ≈37  (18.6-34.9) Neurotensin 15, 60 not yet ≈1000 N.D. determined

[0156] In a separate experiment, the time to peak effect and duration ofaction of CGX-1160 was examined. I.c.v. administration of 100 pmol(approximately 14×ED₅₀) of CGX-1160 showed no activity at 30 minutes,but was 100% protective at 60 minutes, and still showed 50% protectionin animals tested 4 hours following i.c.v. injection (FIG. 20).

[0157] It will be appreciated that the methods and compositions of theinstant invention can be incorporated in the form of a variety ofembodiments, only a few of which are disclosed herein. It will beapparent to the artisan that other embodiments exist and do not departfrom the spirit of the invention. Thus, embodiments described areillustrative and should not be construed as restrictive.

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[0252] PCT Published Application WO 97/12635.

[0253]

1 13 1 16 PRT Conus geographus PEPTIDE (1)..(13) Xaa at residue 1 ispyro-Glu; Xaa at residue 13 is Pro or hydroxy-Pro; Thr at residue 10 ismodified to contain an O-glycan. 1 Xaa Ser Glu Glu Gly Gly Ser Asn AlaThr Lys Lys Xaa Tyr Ile Leu 1 5 10 15 2 16 PRT Artificial SequenceDescription of Artificial SequenceGeneric Contulakin-G formula 2 Xaa XaaXaa Xaa Gly Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Leu 1 5 10 15 3 17DNA Conus geographus misc_feature (1)..(17) n is any nucleotide 3atratnggyt tyttngt 17 4 15 PRT Conus geographus PEPTIDE (9) Xaa atresidue 9 is unknown 4 Ser Glu Glu Gly Gly Ser Asn Ala Xaa Lys Lys ProTyr Ile Leu 1 5 10 15 5 231 DNA Conus geographus CDS (1)..(228) 5 atgcag acg gcc tac tgg gtg atg gtg atg atg atg gtg tgg att gca 48 Met GlnThr Ala Tyr Trp Val Met Val Met Met Met Val Trp Ile Ala 1 5 10 15 gcccct ctg tct gaa ggt ggt aaa ctg aac gat gta att cgg ggt ttg 96 Ala ProLeu Ser Glu Gly Gly Lys Leu Asn Asp Val Ile Arg Gly Leu 20 25 30 gtg ccagac gac ata acc cca cag ctc atg ttg gga agt ctg att tcc 144 Val Pro AspAsp Ile Thr Pro Gln Leu Met Leu Gly Ser Leu Ile Ser 35 40 45 cgt cgt caatcg gaa gag ggt ggt tca aat gca acc aag aaa ccc tat 192 Arg Arg Gln SerGlu Glu Gly Gly Ser Asn Ala Thr Lys Lys Pro Tyr 50 55 60 att cta agg gccagc gac cag gtt gca tct ggg cca tag 231 Ile Leu Arg Ala Ser Asp Gln ValAla Ser Gly Pro 65 70 75 6 76 PRT Conus geographus 6 Met Gln Thr Ala TyrTrp Val Met Val Met Met Met Val Trp Ile Ala 1 5 10 15 Ala Pro Leu SerGlu Gly Gly Lys Leu Asn Asp Val Ile Arg Gly Leu 20 25 30 Val Pro Asp AspIle Thr Pro Gln Leu Met Leu Gly Ser Leu Ile Ser 35 40 45 Arg Arg Gln SerGlu Glu Gly Gly Ser Asn Ala Thr Lys Lys Pro Tyr 50 55 60 Ile Leu Arg AlaSer Asp Gln Val Ala Ser Gly Pro 65 70 75 7 16 PRT Conus geographusPEPTIDE (1)..(10) Xaa at residue 1 is pyro-Glu; Thr at residue 10contains an O-glycan. 7 Xaa Ser Glu Glu Gly Gly Glu Asn Ala Thr Lys LysPro Tyr Ile Leu 1 5 10 15 8 13 PRT Bos sp. PEPTIDE (1) Xaa at residue 1is pyro-Glu. 8 Xaa Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu 1 510 9 6 PRT porcine 9 Lys Ile Pro Tyr Ile Leu 1 5 10 8 PRT Xenopus laevis10 Gln Gly Lys Arg Pro Trp Ile Leu 1 5 11 25 PRT Homo sapiens 11 Met LeuThr Lys Phe Glu Thr Lys Ser Ala Arg Val Lys Gly Leu Ser 1 5 10 15 PheHis Pro Lys Arg Pro Trp Ile Leu 20 25 12 17 PRT Vespula maculifrons 12Thr Ala Thr Thr Arg Arg Arg Gly Arg Pro Pro Gly Phe Ser Pro Phe 1 5 1015 Arg 13 9 PRT Homo sapiens 13 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 5

What is claimed is:
 1. A method for treating pain in an individual whichcomprises administering an analgesic effective amount of an active agentto an individual in need of pain treatment, said active agent comprisescontulakin-G which comprises the amino acid sequenceXaa₁-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Xaa₂-Tyr-Ile-Leu (SEQID NO:1), where Xaa₁ is pyro-Glu, Xaa₂ is proline or hydroxyproline andThr₁₀ is modified to contain an O-glycan.
 2. The method of claim 1,wherein said pain is acute pain.
 3. The method of claim 2, wherein saidacute pain is post-trauma.
 4. The method of claim 1, wherein said painis chronic pain.
 5. The method of claim 4, wherein said chronic painresults from cancer.
 6. The method of claim 4, wherein said chronic painis neuropathic pain.
 7. The method of claim 4, wherein said chronic painis inflammatory.
 8. The method of claim 1, wherein the active agent isadministered using a delivery system selected from the group consistingof infusion, pump delivery, bioerodable polymer delivery,microencapsulated cell delivery, injection and macroencapsulated celldelivery.
 9. The method of claim 8, wherein administration is into thecentral nervous system.
 10. The method of claim 9, wherein the centralnervous system is selected from the group consisting of the intrathecalspace, the brain ventricles and the brain parenchyma.
 11. The method ofclaim 8, wherein the administration is selected from the groupconsisting of subcutaneous, intravenous, intra-arterial andintramuscular.
 12. The method of claim 1, wherein the glycan isGal(β1→3)GalNAc(α1→).
 13. The method of claim 1, wherein the glycan hasthe structure

wherein R₁ is Thr; X is 0; R₂ is OH, NH₂, NHSO₃Na, NHAc, O-sulphate,O-phosphate, or O-glycan; R₃ is H, SO₃, PO₃, acetyl, sialic acid ormonosaccharide; R₄ is H, SO₃, PO₃, acetyl or monosaccharide; R₅ is OH,NH₂, NHSO₃Na, NHAc, O-sulphate, O-phosphate, O-monosaccharide or,O-acetyl; R₆ is H, SO₃, PO₃, acetyl or monosaccharide; R₇ is H, SO₃,PO₃, acetyl or monosaccharide; R₈ is H, SO₃, PO₃, acetyl ormonosaccharide; n is 0-4 and m is 1-4.
 14. A method for treating pain inan individual which comprises administering an analgesic effectiveamount of an active agent to an individual in need of pain treatment,said active agent is selected from the group consisting of (a) a genericcontulakin-G having the following general formulaXaa₁-Xaa₂-Xaa₃-Xaa₃-Gly-Gly-Xaa₂-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Ile-Leu(SEQ ID NO:2), where Xaa₁ is pyro-Glu, Glu, Gln or γ-carboxy-Glu; Xaa₂is Ser, Thr or S-glycan modified Cys; Xaa₃ is Glu or γ-carboxy-Glu; Xaa₄is Asn, N-glycan modified Asn or S-glycan modified Cys; Xaa₅ is Ala orGly; Xaa₆ is Thr, Ser, S-glycan modified Cys, Tyr or any hydroxycontaining unnatural amino acid; Xaa₇ is Lys, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine orany unnatural basic amino acid; Xaa₈ is Ala, Gly, Lys, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine, anyunnatural basic amino acid or X-Lys where X is (CH₂)_(n), phenyl,—(CH₂)_(m)—(CH═CH)—(CH₂)_(m)H or —(CH₂)_(m)—(C≡C)—(CH₂)_(m)H in which nis 1-4 and m is 0-2; Xaa₉ is Pro or hydroxy-Pro; and Xaa₁₀ is Tyr,mono-iodo-Tyr, di-iodo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, Trp,D-Trp, bromo-Trp, bromo-D-Trp, chloro-Trp, chloro-D-Trp, Phe, L-neo-Trp,or any unnatural aromatic amino acid, with the proviso that the genericcontulakin-G is not un-glycosylated contulakin-G; (b) a genericcontulakin-G of (a) which is modified to contain an O-glycan, anS-glycan or an N-glycan; (c) a contulakin-G analog which comprises anN-terminal truncation of from 1 to 9 amino acids of the genericcontulakin-G of (a); (d) a contulakin-G analog of (c), wherein aSer-O-glycan, Thr-O-glycan or Cys-S-glycan is substituted for the aminoacid residue at the truncated N-terminus; (e) a contulakin-G analog,wherein a Ser-O-glycan, Thr-O-glycan or Cys-S-glycan is substituted fora residue at positions 1-9 of the generic contulakin-G of (a); and (f) acontulakin-G analog which comprises an N-terminal truncation of 10 aminoacids of the generic contulakin-G of (a) which is further modified tocontain a Lys-N-glycan at residue 11 of the generic contulakin-G. 15.The method of claim 14, wherein said pain is acute pain.
 16. The methodof claim 15, wherein said acute pain is post-trauma.
 17. The method ofclaim 14, wherein said pain is chronic pain.
 18. The method of claim 17,wherein said chronic pain results from cancer.
 19. The method of claim17, wherein said chronic pain is neuropathic pain.
 20. The method ofclaim 17, wherein said chronic pain is inflammatory.
 21. The method ofclaim 14, wherein the active agent is administered using a deliverysystem selected from the group consisting of infusion, pump delivery,bioerodable polymer delivery, microencapsulated cell delivery, injectionand macroencapsulated cell delivery.
 22. The method of claim 21, whereinadministration is into the central nervous system.
 23. The method ofclaim 22, wherein the central nervous system is selected from the groupconsisting of the intrathecal space, the brain ventricles and the brainparenchyma.
 24. The method of claim 21, wherein the administration isselected from the group consisting of subcutaneous, intravenous,intra-arterial and intramuscular.
 25. The method of claim 14, whereinthe glycan is Gal(β1→3)GalNAc(α1→).
 26. The method of claim 14, whereinthe glycan has the structure

wherein R₁ is Thr, Ser, Cys, Asn or Lys; X is 0 when R is Thr or Ser, orX is S when R₁ is Cys or X is N when R₁ is Asn or Lys; R₂ is OH, NH₂,NHSO₃Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R₃ is H, SO₃, PO₃,acetyl, sialic acid or monosaccharide; R₄ is H, SO₃, PO₃, acetyl ormonosaccharide; R₅ is OH, NH₂, NHSO₃Na, NHAc, O-sulphate, O-phosphate,O-monosaccharide or, O-acetyl; R₆ is H, SO₃, PO₃, acetyl ormonosaccharide; R₇ is H, SO₃, PO₃, acetyl or monosaccharide; R₈ is H,SO₃, PO₃, acetyl or monosaccharide; n is 0-4 and m is 1-4.